Antibodies specific for platelet-activating factor acetylhydrolase

ABSTRACT

The present invention provides purified and isolated polynucleotide sequences encoding human plasma platelet-activating factor acetylhydrolase. Also provided are materials and methods for the recombinant production of platelet-activating factor acetylhydrolase products which are expected to be useful in regulating pathological inflammatory events.

This application is a continuation-in-part of co-pending U.S. patentapplication Ser. No. 08/318,905 filed Oct. 6, 1994, which in turn is acontinuation-in-part of co-pending U.S. patent application Ser. No.08/133,803 filed Oct. 6, 1993 now abandoned.

FIELD OF THE INVENTION

The present invention relates generally to platelet-activating factoracetylhydrolase and more specifically to novel purified and isolatedpolynucleotides encoding human plasma platelet-activating factoracetylhydrolase, to the platelet-activating factor acetylhydrolaseproducts encoded by the polynucleotides, to materials and methods forthe recombinant production of platelet-activating factor acetylhydrolaseproducts and to antibody substances specific for platelet-activatingfactor acetylhydrolase.

BACKGROUND

Platelet-activating factor (PAF) is a biologically active phospholipidsynthesized by various cell types. In vivo and at normal concentrationsof 10⁻¹⁰ to 10⁻⁹ M, PAF activates target cells such as platelets andneutrophils by binding to specific G protein-coupled cell surfacereceptors Venable et al., J. Lipid Res., 34: 691-701 (1993)!. PAF hasthe structure 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine. Foroptimal biological activity, the sn-1 position of the PAF glycerolbackbone must be in an ether linkage with a fatty alcohol and the sn-3position must have a phosphocholine head group.

PAF functions in normal physiological processes (e.g., inflammation,hemostasis and parturition) and is implicated in pathologicalinflammatory responses (e.g., asthma, anaphylaxis, septic shock andarthritis) Venable et al., supra, and Lindsberg et al., Ann. Neurol.,30: 117-129 (1991)!. The likelihood of PAF involvement in pathologicalresponses has prompted attempts to modulate the activity of PAF and themajor focus of these attempts has been the development of antagonists ofPAF activity which interfere with binding of PAF to cell surfacereceptors. See, for example, Heuer et al., Clin. Exp. Allergy, 22:980-983 (1992).

The synthesis and secretion of PAF as well as its degradation andclearance appear to be tightly controlled. To the extent thatpathological inflammatory actions of PAF result from a failure of PAFregulatory mechanisms giving rise to excessive production, inappropriateproduction or lack of degradation, an alternative means of modulatingthe activity of PAF would involve mimicing or augmenting the naturalprocess by which resolution of inflammation occurs. MacrophagesStafforini et al., J. Biol. Chem., 265(17): 9682-9687 (1990)!,hepatocytes and the human hepatoma cell line HepG2 Satoh et al., J.Clin. Invest., 87: 476-481 (1991) and Tarbet et al., J. Biol. Chem.,266(25): 16667-16673 (1991)! have been reported to release an enzymaticactivity, PAF acetylhydrolase (PAF-AH), that inactivates PAF. Inaddition to inactivating PAF, PAF-AH also inactivates oxidativelyfragmented phospholipids such as products of the arachidonic acidcascade that mediate inflammation. See, Stremler et al., J. Biol. Chem.,266(17): 11095-11103 (1991). The inactivation of PAF by PAF-AH occursprimarily by hydrolysis of the PAF sn-2 acetyl group and PAF-AHmetabolizes oxidatively fragmented phospholipids by removing sn-2 acylgroups. Two types of PAF-AH have been identified: cytoplasmic formsfound in a variety of cell types and tissues such as endothelial cellsand erythrocytes, and an extracellular form found in plasma and serum.Plasma PAF-AH does not hydrolyze intact phospholipids except for PAF andthis substrate specificity allows the enzyme to circulate in vivo in afully active state without adverse effects. The plasma PAF-AH appears toaccount for all of the PAF degradation in human blood ex vivo Stafforiniet al., J. Biol. Chem., 262(9): 4223-4230 (1987)!.

While the cytoplasmic and plasma forms of PAF-AH appear to haveidentical substrate specificity, plasma PAF-AH has biochemicalcharacteristics which distinguish it from cytoplasmic PAF-AH and fromother characterized lipases. Specifically, plasma PAF-AH is associatedwith lipoprotein particles, is inhibited by diisopropyl fluorophosphate,is not affected by calcium ions, is relatively insensitive toproteolysis, and has an apparent molecular weight of 43,000 daltons.See, Stafforini et al. (1987), supra. The same Stafforini et al. articledescribes a procedure for partial purification of PAF-AH from humanplasma and the amino acid composition of the plasma material obtained byuse of the procedure. Cytoplasmic PAF-AH has been purified fromerythrocytes as reported in Stafforini et al., J. Biol. Chem., 268(6):3857-3865 (1993) and ten amino terminal residues of cytoplasmic PAF-AHare also described in the article. Hattori et al., J. Biol. Chem.,268(25): 18748-18753 (1993) describes the purification of cytoplasmicPAF-AH from bovine brain. Subsequent to filing of the parent applicationhereto the nucleotide sequence of bovine brain cytoplasmic PAF-AH waspublished in Hattori et al., J. Biol. Chem., 269(237): 23150-23155(1994). On Jan. 5, 1995, three months after the filing date of theparent application hereto, a nucleotide sequence for a lipoproteinassociated phospholipase A₂ (Lp-PLA₂) was published in SmithklineBeecham PLC Patent Cooperation Treaty (PCT) International PublicationNo. WO 95/00649. The nucleotide sequence of the Lp-PLA₂ differs at oneposition when compared to the nucleotide sequence of the PAF-AH of thepresent invention. The nucleotide difference (corresponding to position1297 of SEQ ID NO:7) results in an amino acid difference between theenzymes encoded by the polynucleotides. The amino acid at position 379of SEQ ID NO:8 is a valine while the amino acid at the correspondingposition in Lp-PLA₂ is an alanine. In addition, the nucleotide sequenceof the PAF-AH of the present invention includes 124 bases at the 5' endand twenty bases at the 3' end not present in the Lp-PLA₂ sequence.Three months later, on Apr. 10, 1995, a Lp-PLA₂ sequence was depositedin GenBank under Accession No. U24577 which differs at eleven positionswhen compared to the nucleotide sequence of the PAF-AH of the presentinvention. The nucleotide differences (corresponding to position 79, 81,84, 85, 86, 121, 122, 904, 905, 911, 983 and 1327 of SEQ ID NO:7)results in four amino acid differences between the enzymes encoded bythe polynucleotides. The amino acids at positions 249, 250, 274 and 389of SEQ ID NO:8 are lysine, aspartic acid, phenylalanine and leucine,respectively, while the respective amino acid at the correspondingpositions in the GenBank sequence are isoleucine, arginine, leucine andserine.

The recombinant production of PAF-AH would make possible the use ofexogenous PAF-AH to mimic or augment normal processes of resolution ofinflammation in vivo. The administration of PAF-AH would provide aphysiological advantage over administration of PAF receptor antagonistsbecause PAF-AH is a product normally found in plasma. Moreover, becausePAF receptor antagonists which are structurally related to PAF inhibitnative PAF-AH activity, the desirable metabolism of PAF and ofoxidatively fragmented phospholipids is thereby prevented. Thus, theinhibition of PAF-AH activity by PAF receptor antagonists counteractsthe competitive blockade of the PAF receptor by the antagonists. See,Stremler et al., supra. In addition, in locations of acute inflammation,for example, the release of oxidants results in inactivation of thenative PAF-AH enzyme in turn resulting in elevated local levels of PAFand PAF-like compounds which would compete with any exogenouslyadministed PAF receptor antagonist for binding to the PAF receptor. Incontrast, treatment with recombinant PAF-AH would augment endogenousPAF-AH activity and compensate for any inactivated endogenous enzyme.

There thus exists a need in the art to identify and isolatepolynucleotide sequences encoding human plasma PAF-AH, to developmaterials and methods useful for the recombinant production of PAF-AHand to generate reagents for the detection of PAF-AH in plasma.

SUMMARY OF THE INVENTION

The present invention provides novel purified and isolatedpolynucleotides (i.e., DNA and RNA both sense and antisense strands)encoding human plasma PAF-AH or enzymatically active fragments thereof.Preferred DNA sequences of the invention include genomic and cDNAsequences as well as wholly or partially chemically synthesized DNAsequences. The DNA sequence encoding PAF-AH that is set out in SEQ IDNO:7 and DNA sequences which hybridize to the noncoding strand thereofunder standard stringent conditions or which would hybridize but for theredundancy of the genetic code, are contemplated by the invention. Alsocontemplated by the invention are biological replicas (i.e., copies ofisolated DNA sequences made in vivo or in vitro) of DNA sequences of theinvention. Autonomously replicating recombinant constructions such asplasmid and viral DNA vectors incorporating PAF-AH sequences andespecially vectors wherein DNA encoding PAF-AH is operatively linked toan endogenous or exogenous expression control DNA sequence and atranscription terminator are also provided.

According to another aspect of the invention, procaryotic or eucaryotichost cells are stably transformed with DNA sequences of the invention ina manner allowing the desired PAF-AH to be expressed therein. Host cellsexpressing PAF-AH products can serve a variety of useful purposes. Suchcells constitute a valuable source of immunogen for the development ofantibody substances specifically immunoreactive with PAF-AH. Host cellsof the invention are conspicuously useful in methods for the large scaleproduction of PAF-AH wherein the cells are grown in a suitable culturemedium and the desired polypeptide products are isolated from the cellsor from the medium in which the cells are grown by, for example,immunoaffinity purification.

A non-immunological method contemplated by the invention for purifyingPAF-AH from plasma includes the following steps: (a) isolating lowdensity lipoprotein particles; (b) solubilizing said low densitylipoprotein particles in a buffer comprising 10 mM CHAPS to generate afirst PAF-AH enzyme solution; (c) applying said first PAF-AH enzymesolution to a DEAE anion exchange column; (d) washing said DEAE anionexchange column using an approximately pH 7.5 buffer comprising 1 mMCHAPS; (e) eluting PAF-AH enzyme from said DEAE anion exchange column infractions using approximately pH 7.5 buffers comprising a gradient of 0to 0.5M NaCl; (f) pooling fractions eluted from said DEAE anion exchangecolumn having PAF-AH enzymatic activity; (g) adjusting said pooled,active fractions from said DEAE anion exchange column to 10 mM CHAPS togenerate a second PAF-AH enzyme solution; (h) applying said secondPAF-AH enzyme solution to a blue dye ligand affinity column; (i) elutingPAF-AH enzyme from said blue dye ligand affinity column using a buffercomprising 10 mM CHAPS and a chaotropic salt; (j) applying the eluatefrom said blue dye ligand affinity column to a Cu ligand affinitycolumn; (k) eluting PAF-AH enzyme from said Cu ligand affinity columnusing a buffer comprising 10 mM CHAPS and imidazole; (l) subjecting theeluate from said Cu ligand affinity column to SDS-PAGE; and (m)isolating the approximately 44 kDa PAF-AH enzyme from theSDS-polyacrylamide gel. Preferably, the buffer of step (b) is 25 mMTris-HCl, 10 mM CHAPS, pH 7.5; the buffer of step (d) is 25 mM Tris-HCl,1 mM CHAPS; the column of step (h) is a Blue Sepharose Fast Flow column;the buffer of step (i) is 25 mM Tris-HCl, 10 mM CHAPS, 0.5M KSCN, pH7.5; the column of step (j) is a Cu Chelating Sepharose column; and thebuffer of step (k) is 25 mM Tris-HCl, 10 mM CHAPS, 0.5M NaCl, 50 mMimidazole at a pH in a range of about pH 7.5-8.0.

A method contemplated by the invention for purifyingenzymatically-active PAF-AH from E. coli producing PAF-AH includes thesteps of: (a) preparing a centrifugation supernatant from lysed E. coliproducing PAF-AH enzyme; (b) applying said centrifugation supernatant toa blue dye ligand affinity column; (c) eluting PAF-AH enzyme from saidblue dye ligand affinity column using a buffer comprising 10 mM CHAPSand a chaotropic salt; (d) applying said eluate from said blue dyeligand affinity column to a Cu ligand affinity column; and (e) elutingPAF-AH enzyme from said Cu ligand affinity column using a buffercomprising 10 mM CHAPS and imidazole. Preferably, the column of step (b)is a Blue Sepharose Fast Flow column; the buffer of step (c) is 25 mMTris-HCl, 10 mM CHAPS, 0.5M KSCN, pH. 7.5; the column of step (d) is aCu Chelating Sepharose column; and the buffer of step (e) is 25 mMTris-HCl, 10 mM CHAPS, 0.5M NaCl, 100 mM imidazole, pH 7.5.

Another method contemplated by the invention for purifyingenzymatically-active PAF-AH from E. coli producing PAF-AH includes thesteps of: (a) preparing a centrifugation supernatant from lysed E. coliproducing PAF-AH enzyme; (b) diluting said centrifugation supernatant ina low pH buffer comprising 10 mM CHAPS; (c) applying said dilutedcentrifugation supernatant to a cation exchange column equilibrated atabout pH 7.5; (d) eluting PAF-AH enzyme from said cation exchange columnusing 1M salt; (e) raising the pH of said eluate from said cationexhange column and adjusting the salt concentration of said eluate toabout 0.5M salt; (f) applying said adjusted eluate from said cationexchange column to a blue dye ligand affinity column; (g) eluting PAF-AHenzyme from said blue dye ligand affinity column using a buffercomprising about 2M to about 3M salt; and (h) dialyzing said eluate fromsaid blue dye ligand affinity column using a buffer comprising about0.1% Tween. Preferably, the buffer of step (b) is 25 mM MES, 10 mMCHAPS, 1 mM EDTA, pH 4.9; the column of step (c) is an S sepharosecolumn equilibrated in 25 mM MES, 10 mM CHAPS, 1 mM EDTA, 50 mM NaCl, pH5.5; PAF-AH is eluted in step (d) using 1 mM NaCl; the pH of the eluatein step (e) is adjusted to pH 7.5 using 2M Tris base; the column in step(f) is a sepharose column; the buffer in step (g) is 25 mM Tris, 10 mMCHAPS, 3M NaCl, 1 mM EDTA, pH 7.5; and the buffer in step (h) is 25 mMTris, 0.5M NaCl, 0.1% Tween 80, pH 7.5.

Still another method contemplated by the invention for purifyingenzymatically-active PAF-AH from E. coli includes the steps of: (a)preparing an E. coli extract which yields solubilized PAF-AH supernatantafter lysis in a buffer containing CHAPS; (b) dilution of the saidsupernatant and application to a anion exchange column equilibrated atabout pH 8.0; (c) eluting PAF-AH enzyme from said anion exchange column;(d) applying said adjusted eluate from said anion exchange column to ablue dye ligand affinity column; (e) eluting the said blue dye ligandaffinity column using a buffer comprising 3.0M salt; (f) dilution of theblue dye eluate into a suitable buffer for performing hydroxylapatitechromatography; (g) performing hydroxylapatite chromatography wherewashing and elution is accomplished using buffers (with or withoutCHAPS); (h) diluting said hydroxylapatite eluate to an appropriate saltconcentration for cation exchange chromatography; (i) applying saiddiluted hydroxylapatite eluate to a cation exchange column at a pHranging between approximately 6.0 to 7.0; (j) elution of PAF-AH fromsaid cation exchange column with a suitable formulation buffer; (k)performing cation exchange chromatography in the cold; and (l)formulation of PAF-AH in liquid or frozen form in the absence of CHAPS.

Preferably in step (a) above the lysis buffer is 25 mM Tris, 100 mMNaCl, 1 mM EDTA, 20 mM CHAPS, pH 8.0; in step (b) the dilution of thesupernatant for anion exchange chromatography is 3-4 fold into 25 mMTris, 1 mM EDTA, 10 mM CHAPS, pH 8.0 and the column is a Q-Sepharosecolumn equilibrated with 25 mM Tris, 1 mM EDTA, 50 mM NaCl, 10 mM CHAPS,pH 8.0; in step (c) the anion exchange column is eluted using 25 mMTris, 1 mM EDTA, 350 mM NaCl, 10 mM CHAPS, pH 8.0; in step (d) theeluate from step (c) is applied directly onto a blue dye affinitycolumn; in step (e) the column is eluted with 3M NaCl, 10 mM CHAPS, 25mM Tris, pH 8.0 buffer; in step (f) dilution of the blue dye eluate forhydroxylapatite chromatography is accomplished by dilution into 10 mMsodium phosphate, 100 mM NaCl, 10 mM CHAPS, pH 6.2; in step (g)hydroxylapatite chromatography is accomplished using a hydroxylapatitecolumn equilibrated with 10 mM sodium phosphate, 100 mM NaCl, 10 mMCHAPS and elution is accomplished using 50 mM sodium phosphate, 100 mMNaCl (with or without) 10 mM CHAPS, pH 7.5; in step (h) dilution of saidhydroxylapatite eluate for cation exchange chromatography isaccomplished by dilution into a buffer ranging in pH from approximately6.0 to 7.0 comprising sodium phosphate (with or without CHAPS); in step(i) a S Sepharose column is equilibrated with 50 mM sodium phosphate,(with or without) 10 mM CHAPS, pH 6.8; in step (j) elution isaccomplished with a suitable formulation buffer such as potassiumphosphate 50 mM, 12.5 mM aspartic acid, 125 mM NaCl, pH 7.5 containing0.01% Tween-80; and in step (k) cation exchange chromatrography isaccomplished at 2°-8° C. Examples of suitable formulation buffers foruse in step (l) which stabilize PAF-AH include 50 mM potassiumphosphate, 12.5 mM Aspartic acid, 125 mM NaCl pH 7.4 (approximately,with and without the addition of Tween-80 and or Pluronic F68) or 25 mMpotassium phosphate buffer containing (at least) 125 mM NaCl, 25 mMarginine and 0.01% Tween-80 (with or without Pluronic F68 atapproximately 0.1 and 0.5%).

PAF-AH products may be obtained as isolates from natural cell sources ormay be chemically synthesized, but are preferably produced byrecombinant procedures involving procaryotic or eucaryotic host cells ofthe invention. PAF-AH products having part or all of the amino acidsequence set out in SEQ ID NO:8 are contemplated. The use of mammalianhost cells is expected to provide for such post-translationalmodifications (e.g., myristolation, glycosylation, truncation,lipidation and tyrosine, serine or threonine phosphorylation) as may beneeded to confer optimal biological activity on recombinant expressionproducts of the invention. PAF-AH products of the invention may be fulllength polypeptides, fragments or variants. Variants may comprise PAF-AHanalogs wherein one or more of the specified (i.e., naturally encoded)amino acids is deleted or replaced or wherein one or more nonspecifiedamino acids are added: (1) without loss of one or more of the enzymaticactivities or immunological characteristics specific to PAF-AH; or (2)with specific disablement of a particular biological activity of PAF-AH.Proteins or other molecules that bind to PAF-AH may be used to modulateits activity.

Also comprehended by the present invention are antibody substances(e.g., monoclonal and polyclonal antibodies, single chain antibodies,chimeric antibodies, CDR-grafted antibodies and the like) and otherbinding proteins specific for PAF-AH. Specifically illustrating bindingproteins of the invention are the monoclonal antibodies produced byhybridomas 90G11D and 90F2D which were deposited with the American TypeCulture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852 onSep. 30, 1994 and were respectively assigned Accession Nos. HB 11724 andHB 11725. Also illustrating binding proteins of the invention is themonoclonal antibody produced by hybridoma 143A which was deposited withthe ATCC on Jun. 1, 1995 and assigned Accession No. HB 11900. Proteinsor other molecules (e.g., lipids or small molecules) which specificallybind to PAF-AH can be identified using PAF-AH isolated from plasma,recombinant PAF-AH, PAF-AH variants or cells expressing such products.Binding proteins are useful, in turn, in compositions for immunizationas well as for purifying PAF-AH, and are useful for detection orquantification of PAF-AH in fluid and tissue samples by knownimmunological procedures. Anti-idiotypic antibodies specific forPAF-AH-specific antibody substances are also contemplated.

The scientific value of the information contributed through thedisclosures of DNA and amino acid sequences of the present invention ismanifest. As one series of examples, knowledge of the sequence of a cDNAfor PAF-AH makes possible the isolation by DNA/DNA hybridization ofgenomic DNA sequences encoding PAF-AH and specifying PAF-AH expressioncontrol regulatory sequences such as promoters, operators and the like.DNA/DNA hybridization procedures carried out with DNA sequences of theinvention under conditions of stringency standard in the art arelikewise expected to allow the isolation of DNAs encoding allelicvariants of PAF-AH, other structurally related proteins sharing one ormore of the biochemical and/or immunological properties of PAF-AH, andnon-human species proteins homologous to PAF-AH. The DNA sequenceinformation provided by the present invention also makes possible thedevelopment, by homologous recombination or "knockout" strategies see,e.g., Cappechi, Science, 244: 1288-1292 (1989)!, of rodents that fail toexpress a functional PAF-AH enzyme or that express a variant PAF-AHenzyme. Polynucleotides of the invention when suitably labelled areuseful in hybridization assays to detect the capacity of cells tosynthesize PAF-AH. Polynucleotides of the invention may also be thebasis for diagnostic methods useful for identifying a geneticalteration(s) in the PAF-AH locus that underlies a disease state orstates. Also made available by the invention are anti-sensepolynucleotides relevant to regulating expression of PAF-AH by thosecells which ordinarily express the same.

Administration of PAF-AH preparations of the invention to mammaliansubjects, especially humans, for the purpose of amelioratingpathological inflammatory conditions is contemplated. Based onimplication of the involvement of PAF in pathological inflammatoryconditions, the administration of PAF-AH is indicated, for example, intreatment of asthma Miwa et al., J. Clin. Invest., 82: 1983-1991 (1988);Hsieh et al., J. Allergy Clin. Immunol., 91: 650-657 (1993); andYamashita et al., Allergy, 49: 60-63 (1994)!, anaphylaxis Venable etal., supra!, shock Venable et al., supra!, reperfusion injury andcentral nervous system ischemia Lindsberg et al. (1991), supra!,antigen-induced arthritis Zarco et al., Clin. Exp. Immunol., 88: 318-323(1992)!, atherogenesis Handley et al., Drug Dev. Res., 7: 361-375(1986)!, Crohn's disease Denizot et al., Digestive Diseases andSciences, 37(3): 432-437 (1992)!, ischemic bowel necrosis/necrotizingenterocolitis Denizot et al., supra and Caplan et al., Acta Paediatr.,Suppl. 396. 11-17 (1994)!, ulcerative colitis (Denizot et al., supra),ischemic stroke Satoh et al., Stroke, 23: 1090-1092 (1992)!, ischemicbrain injury Lindsberg et al., Stroke, 21: 1452-1457 (1990) andLindsberg et al. (1991), supra!, systemic lupus erythematosus Matsuzakiet al., Clinica Chimica Acta, 210: 139-144 (1992)!, acute pancreatitisKald et a., Pancreas, 8(4): 440-442 (1993)!, septicemia (Kald et al.,supra), acute post streptococcal glomerulonephritis Mezzano et al., J.Am. Soc. Nephrol, 4: 235-242 (1993)!, pulmonary edema resulting fromIL-2 therapy Rabinovici et al., J. Clin. Invest., 89: 1669-1673 (1992)!,allergic inflammation Watanabe et al., Br. J. Pharmacol., 111: 123-130(1994)!, ischemic renal failure Grino et al., Annals of InternalMedicine, 121(5): 345-347 (1994); preterm labor Hoffman et al., Am. J.Obstet. Gynecol., 162(2): 525-528 (1990) and Maki et al., Proc. Natl.Acad. Sci. USA, 85: 728-732 (1988)!; and adult respiratory distresssyndrome Rabinovici et al., J. Appl. Physiol., 74(4): 1791-1802 (1993);Matsumoto et al., Clin. Exp. Pharmacol. Physiol., 19 509-515 (1992); andRodriguez-Roisin et al., J. Clin. Invest., 93: 188-194 (1994)!.

Animal models for many of the foregoing pathological conditions havebeen described in the art. For example, a mouse model for asthma andrhinitis is described in Example 16 herein; a rabbit model for arthritisis described in Zarco et at., supra; rat models for ischemic bowelnecrosis/necrotizing enterocolitis are described in Furukawa et al.,Ped. Res., 34,(2): 237-241 (1993) and Caplan et al., supra; a rabbitmodel for stroke is described in Lindsberg et al., (1990), supra; amouse model for lupus is described in Matsuzaki et al., supra; a ratmodel for acute pancreatitis is described in Kald et al., supra: a ratmodel for pulmonary edema resulting from IL-2 therapy is described inRabinovici et al., supra; a rat model of allergic inflammation isdescribed in Watanabe et al., supra); a canine model of renal allograftis described in Watson et al., Transplantation, 56(4): 1047-1049 (1993);and rat and guinea pig models of adult respiratory distress syndrome arerespectively described in Rabinovici et al., supra. andLellouch-Tubiana, Am. Rev. Respir. Dis., 137: 948-954 (1988).

Specifically contemplated by the invention are PAF-AH compositions foruse in methods for treating a mammal susceptible to or suffering fromPAF-mediated pathological conditions comprising administering PAF-AH tothe mammal in an amount sufficient to supplement endogenous PAF-AHactivity and to inactivate pathological amounts of PAF in the mammal.

Therapeutic/pharmaceutical compositions contemplated by the inventioninclude PAF-AH and a physiologically acceptable diluent or carrier andmay also include other agents having anti-inflammatory effects. Dosageamounts indicated would be sufficient to supplement endogenous PAF-AHactivity and to inactivate pathological amounts of PAF. For generaldosage considerations see Remmington's Pharmaceutical Sciences, 18thEdition, Mack Publishing Co., Easton, Pa. (1990). Dosages will varybetween about 0.1 to about 1000 μg PAF-AH/kg body weight. Therapeuticcompositions of the invention may be administered by various routesdepending on the pathological condition to be treated. For example,administration may be by intraveneous, subcutaneous, oral, suppository,and/or pulmonary routes.

For pathological conditions of the lung, administration of PAF-AH by thepulmonary route is particularly indicated. Contemplated for use inpulmonary administration are a wide range of delivery devices including,for example, nebulizers, metered dose inhalers, and powder inhalers,which are standard in the art. Delivery of various proteins to the lungsand circulatory system by inhalation of aerosol formulations has beendescribed in Adjei et al., Pharm. Res., 7(6): 565-569 (1990) (leuprolideacetate); Braquet et al., J. Cardio. Pharm., 13(Supp. 5): s. 143-146(1989) (endothelin-1); Hubbard et al., Annals of Internal Medicine,111(3), 206-212(1989) (α1-antitrypsin); Smith et al., J. Clin. Invest.,84: 1145-1146(1989) (α-1-proteinase inhibitor); Debs et al., J.Immunol., 140: 3482-3488 (1933) (recombinant gamma interferon and tumornecrosis factor alpha); Patent Cooperation Treaty (PCT) InternationalPublication No. WO 94/20069 published Sep. 15, 1994 (recombinantpegylated granulocyte colony stimulating factor).

BRIEF DESCRIPTION OF THE DRAWING

Numerous other aspects and advantages of the present invention will beapparent upon consideration of the following detailed descriptionthereof, reference being made to the drawing wherein:

FIG. 1 is a photograph of a PVDF membrane containing PAF-AH purifiedfrom human plasma;

FIG. 2 is a graph showing the enzymatic activity of recombinant humanplasma PAF-AH;

FIG. 3 is a schematic drawing depicting recombinant PAF-AH fragments andtheir catalytic activity;

FIG. 4 is a bar graph illustrating blockage of PAF-induced rat footedema by locally administered recombinant PAF-AH of the invention;

FIG. 5 is a bar graph illustrating blockage of PAF-induced rat footedema by intravenously administered PAF-AH;

FIG. 6 is a bar graph showing that PAF-AH blocks PAF-induced edema butnot zymosan A-induced edema;

FIGS. 7A and 7B present dose response results of PAF-AHanti-inflammatory activity in rat food edema;

FIGS. 8A and 8B present results indicating the in vivo efficacy of asingle dose of PAF-AH over time;

FIG. 9 is a line graph representing the pharmacokinetics of PAF-AH inrat circulation; and

FIG. 10 is a bar graph showing the anti-inflammatory effects of PAF-AHin comparison to the lesser effects of PAF antagonists in rat footedema.

DETAILED DESCRIPTION

The following examples illustrate the invention. Example 1 presents anovel method for the purification of PAF-AH from human plasma. Example 2describes amino acid microsequencing of the purified human plasmaPAF-AH. The cloning of a full length cDNA encoding human plasma PAF-AHis described in Example 3. Identification of a putative splice variantof the human plasma PAF-AH gene is described in Example 4. The cloningof genomic sequences encoding human plasma PAF-AH is described inExample 5. Example 6 desribes the cloning of canine, murine, bovine,chicken, rodent and macaque cDNAs homologous to the human plasma PAF-AHcDNA. Example 7 presents the results of an assay evidencing theenzymatic activity of recombinant PAF-AH transiently expressed in COS 7cells. Example 8 describes the expression of human PAF-AH in E. coli, S.cerevisiae and mammalian cells. Example 9 presents protocols forpurification of recombinant PAF-AH from E. coli and assays confirmingits enzymatic activity. Example 10 describes various recombinant PAF-AHproducts including amino acid substitution analogs and amino andcarboxy-truncated products, and describes experiments demonstrating thatnative PAF-AH isolated from plasma is glycosylated. Results of aNorthern blot assay for expression of human plasma PAF-AH RNA in varioustissues and cell lines are presented in Example 11 while results of insitu hybridzation are presented in Example 12. Example 13 describes thedevelopment of monoclonal and polyclonal antibodies specific for humanplasma PAF-AH. Examples 14, 15, 16, 17 and 18 respectively describe thein vivo therapeutic effect of administration of recombinant PAF-AHproducts of the invention on acute inflammation, pleurisy, asthma,necrotizing enterocolitis, and adult respiratory distress syndrome inanimal models. Example 19 presents the results of immunoassays of serumof human patients exhibiting a deficiency in PAF-AH activity anddescribes the identification of a genetic lesion in the patients whichis apparently responsible for the deficiency.

EXAMPLE 1

PAF-AH was purified from human plasma in order to provide material foramino acid sequencing.

A. Optimization of Purification Conditions

Initially, low density lipoprotein (LDL) particles were precipitatedfrom plasma with phosphotungstate and solubilized in 0.1% Tween 20 andsubjected to chromatography on a DEAE column (Pharmacia, Uppsala,Sweden) according to the method of Stafforini et al. (1987), supra, butinconsistent elution of PAF-AH activity from the DEAE column requiredreevaluation of the solubilization and subsequent purificationconditions.

Tween 20, CHAPS (Pierce Chemical Co., Rockford, Ill.) and octylglucoside were evaluated by centrifugation and gel filtrationchromatography for their ability to solubilize LDL particles. CHAPSprovided 25% greater recovery of solubilized activity than Tween 20 and300% greater recovery than octyl glucoside. LDL precipitate solubilizedwith 10 mM CHAPS was then fractionated on a DEAE Sepharose Fast Flowcolumn (an anion exchange column; Pharmacia) with buffer containing 1 mMCHAPS to provide a large pool of partially purified PAF-AH ("the DEAEpool") for evaluation of additional columns.

The DEAE pool was used as starting material to test a variety ofchromatography columns for utility in further purifying the PAF-AHactivity. The columns tested included: Blue Sepharose Fast Flow(Pharmacia), a dye ligand affinity column; S-Sepharose Fast Flow(Pharmacia), a cation exchange column; Cu Chelating Sepharose(Pharmacia), a metal ligand affinity column; Fractogel S (EMSeparations, Gibbstown, N.J.), a cation exchange column; andSephacryl-200 (Pharmacia), a gel filtration column. Thesechromatographic procedures all yielded low, unsatisfactory levels ofpurification when operated in 1 mM CHAPS. Subsequent gel filtrationchromatography on Sephacryl S-200 in 1 mM CHAPS generated anenzymatically active fraction which eluted over a broad size rangerather than the expected 44 kDa approximate size. Taken together, theseresults indicated that the LDL proteins were aggregating in solution.

Different LDL samples were therefore evaluated by analytical gelfiltration chromatography for aggregation of the PAF-AH activity.Samples from the DEAE pool and of freshly solubilized LDL precipitatewere analyzed on Superose 12 (Pharmacia) equilibrated in buffer with 1mM CHAPS. Both samples eluted over a very broad range of molecularweights with most of the activity eluting above 150 kDa. When thesamples were then analyzed on Superose 12 equilibrated with 10 mM CHAPS,the bulk of the activity eluted near 44 kDa as expected for PAF-AHactivity. However, the samples contained some PAF-AH activity in thehigh molecular weight region corresponding to aggregates.

Other samples eluted PAF-AH activity exclusively in the approximately 44kDa range when they were subsequently tested by gel filtration. Thesesamples were an LDL precipitate solubilized in 10 mM CHAPS in thepresence of 0.5M NaCl and a fresh DEAE pool that was adjusted to 10 mMCHAPS after elution from the DEAE column. These data indicate that atleast 10 mM CHAPS is required to maintain non-aggregated PAF-AH.Increase of the CHAPS concentration from 1 mM to 10 mM afterchromatography on DEAE but prior to subsequent chromatographic stepsresulted in dramatic differences in purification. For example, thedegree of PAF-AH purification on S-Sepharose Fast Flow was increasedfrom 2-fold to 10-fold. PAF-AH activity bound the Blue Sepharose FastFlow column irreversibly in 1 mM CHAPS, but the column provided thehighest level of purification in 10 mM CHAPS. The DEAE chromatographywas not improved with prior addition of 10 mM CHAPS.

Chromatography on Cu Chelating Sepharose after the Blue Sepharose FastFlow column concentrated PAF-AH activity 15-fold. It was also determinedthat PAF-AH activity could be recovered from a reducedSDS-polyacrylamide gel, as long as samples were not boiled. The activityof material eluted from the Cu Chelating Sepharose column when subjectedto SDS-polyacrylamide gel electrophoresis coincided with a major proteinband when the gel was silver stained.

B. PAF-AH Purification Protocol

The novel protocol utilized to purify PAF-AH for amino acid sequencingtherefore comprised the following steps which were performed at 4° C.Human plasma was divided into 900 ml aliquots in 1 liter Nalgene bottlesand adjusted to pH 8.6. LDL particles were then precipitated by adding90 ml of 3.85% sodium phosphotungstate followed by 23 ml of 2M MgCl₂.The plasma was then centrifuged for 15 minutes at 3600 g. Pellets wereresuspended in 800 ml of 0.2% sodium citrate. LDL was precipitated againby adding 10 g NaCl and 24 ml of 2M MgCl₂. LDL particles were pelletedby centrifugation for 15 minutes at 3600 g. This wash was repeatedtwice. Pellets were then frozen at -20° C. LDL particles from 5 L ofplasma were resuspended in 5 L of buffer A (25 mM Tris-HCl, 10 mM CHAPS,pH 7.5) and stirred overnight. Solubilized LDL particles werecentrifuged at 3600 g for 1.5 hours. Supernatants were combined andfiltered with Whatman 113 filter paper to remove any remaining solids.Solubilized LDL supernatant was loaded on a DEAE Sepharose Fast Flowcolumn (11 cm×10 cm; 1 L resin volume; 80 ml/minute) equilibrated inbuffer B (25 mM Tris-HCl, 1 mM CHAPS, pH 7.5). The column was washedwith buffer B until absorbance returned to baseline. Protein was elutedwith an 8 L, 0-0.5M NaCl gradient and 480 ml fractions were collected.This step was necessary to obtain binding to the Blue Sepharose FastFlow column below. Fractions were assayed for acetylhydrolase activityessentially by the method described in Example 4.

Active fractions were pooled and sufficient CHAPS was added to make thepool about 10 mM CHAPS. The DEAE pool was loaded overnight at 4ml/minute onto a Blue Sepharose Fast Flow column (5 cm×10 cm; 200 ml bedvolume) equilibrated in buffer A containing 0.5M NaCl. The column waswashed with the equilibration buffer at 16 ml/minute until absorbancereturned to baseline. PAF-AH activity was step eluted with buffer Acontaining 0.5M KSCN (a chaotropic salt) at 16 ml/minute and collectedin 50 ml fractions. This step resulted in greater than 1000-foldpurification. Active fractions were pooled, and the pool was adjusted topH 8.0 with 1M Tris-HCl pH 8.0. The active pool from Blue Sepharose FastFlow chromatography was loaded onto a Cu Chelating Sepharose column (2.5cm×2 cm; 10 ml bed volume; 4 ml/minute) equilibrated in buffer C 25 mMTris-HCl, 10 mM CHAPS, 0.5M NaCl, pH 8.0 (pH 7.5 also worked)!, and thecolumn was washed with 50 ml buffer C. PAF-AH activity was eluted with100 ml 50 mM imidazole in buffer C and collected in 10 ml fractions.Fractions containing PAF-AH activity were pooled and dialyzed againstbuffer A. In addition to providing a 15-fold concentration of PAF-AHactivity, the Cu Chelating Sepharose column gave a small purification.The Cu Chelating Sepharose pool was reduced in 50 mM DTT for 15 minutesat 37° C. and loaded onto a 0.75 mm, 7.5% polyacrylamide gel. Gel sliceswere cut every 0.5 cm and placed in disposable microfuge tubescontaining 200 μl 25 mM Tris-HCl, 10 mM CHAPS, 150 mM NaCl. Slices wereground up and allowed to incubate overnight at 4° C. The supernatant ofeach gel slice was then assayed for PAF-AH activity to determine whichprotein band on SDS-PAGE contained PAF-AH activity. PAF-AH activity wasfound in an approximately 44 kDa band. Protein from a duplicate gel waselectrotransferred to a PVDF membrane (Immobilon-P, Millipore) andstained with Coomassie Blue. A photograph of the PVDF membrane ispresented in FIG. 1.

As presented in Table I below, approximately 200 μg PAF-AH was purified2×10⁶ -fold from 5 L human plasma. In comparison, a 3×10⁴ -foldpurification of PAF-AH activity is described in Stafforini et al.(1987), supra.

                                      TABLE 1                                     __________________________________________________________________________                 Total                                                                             Prot.                                                                            Specific                                                           Activity                                                                          Activity                                                                          Conc.                                                                            Activity                                                                          % Recovery                                            Vol.     (cpm ×                                                                      (cpm ×                                                                      (mg/                                                                             (cpm ×                                                                      of Activity                                                                         Fold Purification                               Sample                                                                              (ml)                                                                             10.sup.6)                                                                         10.sup.9)                                                                         ml)                                                                              10.sup.6)                                                                         Step                                                                             Cum.                                                                             Step                                                                              Cum.                                        __________________________________________________________________________    Plasma                                                                              5000                                                                             23  116 62 0.37                                                                              100                                                                              100                                                                              1   1                                           LDL   4500                                                                             22  97  1.76                                                                             12  84 84 33  33                                          DEAE  4200                                                                             49  207 1.08                                                                             46  212                                                                              178                                                                              3.7 124                                         Blue  165                                                                              881 14  0.02                                                                             54200                                                                             70 126                                                                              1190                                                                              1.5 × 10.sup.5                        Cu    12 12700                                                                             152 0.15                                                                             82200                                                                             104                                                                              131                                                                              1.5 2.2 × 10.sup.5                        SDS-PAGE                                                                            -- --  --  -- --  -- -- ˜10                                                                         2.2 × 10.sup.6                        __________________________________________________________________________

In summary, the following steps were unique and critical for successfulpurification of plasma PAF-AH for microsequencing: (1) solubilizationand chromotography in 10 mM CHAPS, (2) chromatography on a blue ligandaffinity column such as Blue Sepharose Fast Flow, (3) chromatography ona Cu ligand affinity column such as Cu Chelating Sepharose, and (4)elution of PAF-AH from SDS-PAGE.

EXAMPLE 2

For amino acid sequencing, the approximately 44 kDa protein band fromthe PAF-AH-containing PVDF membrane described in Example 1 was excisedand sequenced using an Applied Biosystems 473A Protein sequencer.N-terminal sequence analysis of the approximately 44 kDa protein bandcorresponding to the PAF-AH activity indicated that the band containedtwo major sequences and two minor sequences. The ratio of the two majorsequences was 1:1 and it was therefore difficult to interpret thesequence data.

To distinguish the sequences of the two major proteins which had beenresolved on the SDS gel, a duplicate PVDF membrane containing theapproximately 44 kDa band was cut in half such that the upper part andthe lower part of the membrane were separately subjected to sequencing.

The N-terminal sequence obtained for the lower half of the membrane was:

SEQ ID NO:1 FKDLGEENFKALVLIAF

A search of protein databases revealed this sequence to be a fragment ofhuman serum albumin. The upper half of the same PVDF membrane was alsosequenced and the N-terminal amino acid sequence determined was:

SEQ ID NO:2 IQVLMAAASFGQTKIP

This sequence did not match any protein in the databases searched andwas different from the N-terminal amino acid sequence:

SEQ ID NO:3 MKPLVVFVLGG

which was reported for erythrocyte cytoplasmic PAF-AH in Stafforini etal. (1993), supra. The novel sequence (SEQ ID NO:2) was utilized forcDNA cloning of human plasma PAF-AH as described below in Example 3.

EXAMPLE 3

A full length clone encoding human plasma PAF-AH was isolated from amacrophage cDNA library.

A. Construction of a Macrophage cDNA Library

Poly A⁺ RNA was harvested from peripheral blood monocyte-derivedmacrophages. Double-stranded, blunt-ended cDNA was generated using theInvitrogen Copy Kit (San Diego, Calif.) and BstXI adapters were ligatedto the cDNA prior to insertion into the mammalian expression vector,pRc/CMV (Invitrogen). The resulting plasmids were introduced into E.coli strain XL-1 Blue by electroporation. Transformed bacteria wereplated at a density of approximately 3000 colonies per agarose plate ona total of 978 plates. Plasmid DNA prepared separately from each platewas retained in individual pools and was also combined into larger poolsrepresenting 300,000 clones each.

B. Library Screening by PCR

The macrophage library was screened by the polymerase chain reactionutilizing a degenerate antisense oligonucleotide PCR primer based on thenovel N-terminal amino acid sequence described in Example 2. Thesequence of the primer is set out below in IUPAC nomenclature and where"I" is an inosine.

SEQ ID NO:4 5' ACATGAATTCGGIATCYTRIGTYTGICCRAA 3'

The codon choice tables of Wada et al., Nuc. Acids Res., 19S: 1981-1986(1991) were used to select nucleotides at the third position of eachcodon of the primer. The primer was used in combination with a primerspecific for either the SP6 or T7 promoter sequences, both of whichflank the cloning site of pRc/CMV, to screen the macrophage librarypools of 300,000 clones. All PCR reactions contained 100 ng of templatecDNA, 1 μg of each primer, 0.125 mM of each dNTP, 10 mM Tris-HCl pH 8.4,50 mM MgCl₂ and 2.5 units of Taq polymerase. An initial denaturationstep of 94° C. for four minutes was followed by 30 cycles ofamplification of 1 minute at 94° C., 1 minute at 60° C. and 2 minutes at72° C. The resulting PCR product was cloned into pBluescriptSK-(Stratagene, La Jolla, Calif.) and its nucleotide sequence determinedby the dideoxy chain termination method. The PCR product contained thesequence predicted by the novel peptide sequence and corresponds tonucleotides 1 to 331 of SEQ ID NO:7.

The PCR primers set out below, which are specific for the cloned PCRfragment described above, were then designed for identifying a fulllength clone.

Sense Primer (SEQ ID NO:5) 5' TATTTCTAGAAGTGTGGTGGAACTCGCTGG 3'

Antisense Primer (SEQ ID NO:6) 5° C.GATGAATTCAGCTTGCAGCAGCCATCAGTAC 3'

PCR reactions utilizing the primers were performed as described above tofirst screen the cDNA pools of 300,000 clones and then the appropriatesubset of the smaller pools of 3000 clones. Three pools of 3000 cloneswhich produced a PCR product of the expected size were then used totransform bacteria.

C. Library Screening by Hybridization

DNA from the transformed bacteria was subsequently screened byhybridization using the original cloned PCR fragment as a probe.Colonies were blotted onto nitrocellulose and prehybridized andhybridized in 50% formamide, 0.75M sodium chloride, 0.075M sodiumcitrate, 0.05M sodium phosphate pH 6.5, 1% polyvinyl pyrolidine, 1%Ficoll, 1% bovine serum albumin and 50 ng/ml sonicated salmon sperm DNA.The hybridization probe was labeled by random hexamer priming. Afterovernight hybridization at 42° C., blots were washed extensively in0.03M sodium chloride, 3 mM sodium citrate, 0. 1% SDS at 42° C. Thenucleotide sequence of 10 hybridizing clones was determined. One of theclones, clone sAH 406-3, contained the sequence predicted by theoriginal peptide sequence of the PAF-AH activity purified from humanplasma. The DNA and deduced amino acid sequences of the human plasmaPAF-AH are set out in SEQ ID NOs: 7 and 8, respectively.

Clone sAH 406-3 contains a 1.52 kb insert with an open reading framethat encodes a predicted protein of 441 amino acids. At the aminoterminus, a relatively hydrophobic segment of 41 residues precedes theN-terminal amino acid (the isoleucine at position 42 of SEQ ID NO:8)identified by protein microsequencing. The encoded protein may thus haveeither a long signal sequence or a signal sequence plus an additionalpeptide that is cleaved to yield the mature functional enzyme. Thepresence of a signal sequence is one characteristic of secretedproteins. In addition, the protein encoded by clone sAH 406-3 includesthe consensus GxSxG motif (amino acids 271-275 of SEQ ID NO:8) that isbelieved to contain the active site serine of all known mammalianlipases, microbial lipases and serine proteases. See Chapus et al.,Biochimie, 70: 1223-1224 (1988) and Brenner, Nature, 334: 528-530(1988).

Table 2 below is a comparison of the amino acid composition of the humanplasma PAF-AH of the invention as predicted from SEQ ID NO:8 and theamino acid composition of the purportedly purified material described byStafforini et al. (1987), supra.

                  TABLE 2                                                         ______________________________________                                                   Clone sAH 406-3                                                                         Stafforini et al.                                        ______________________________________                                        Ala          26          24                                                   Asp & Asn    48          37                                                   Cys           5          14                                                   Glu & Gln    36          42                                                   Phe          22          12                                                   Gly          29          58                                                   His          13          24                                                   Ile          31          17                                                   Lys          26          50                                                   Leu          40          26                                                   Met          10           7                                                   Pro          15          11                                                   Arg          18          16                                                   Ser          27          36                                                   Thr          20          15                                                   Val          13          14                                                   Trp           7          Not determined                                       Tyr          14          13                                                   ______________________________________                                    

The amino acid composition of the mature form of the human plasma PAF-AHof the invention and the amino acid composition of the previouslypurified material that was purportedly the human plasma PAF-AH areclearly distinct.

When alignment of the Hattori et al., supra nucleotide and deduced aminoacid sequences of bovine brain cytoplasmic PAF-AH with the nucleotideand amino acid sequences of the human plasma PAF-AH of the invention wasattempted, no significant structural similarity in the sequences wasobserved.

EXAMPLE 4

A putative splice variant of the human PAF-AH gene was detected when PCRwas performed on macrophage and stimulated PBMC cDNA using primers thathybridized to the 5' untranslated region (nucleotides 31 to 52 of SEQ IDNO:7) and the region spanning the translation termination codon at the3' end of the PAF-AH cDNA (nucleotides 1465 to 1487 of SEQ ID NO:7). ThePCR reactions yielded two bands on a gel, one corresponding to theexpected size of the PAF-AH cDNA of Example 3 and the other was about100 bp shorter. Sequencing of both bands revealed that the larger bandwas the PAF-AH cDNA of Example 3 while the shorter band lacked exon 2(Example 5 below) of the PAF-AH sequence which encodes the putativesignal and pro-peptide sequences of plasma PAF-AH. The predictedcatalytic triad and all cysteines were present in the shorter clone,therefore the biochemical activity of the protein encoded by the cloneis likely to match that of the plasma enzyme.

To begin to assess the biological relevance of the PAF-AH splice variantthat is predicted to encode a cytoplasmically active enzyme, therelative abundance of the two forms in blood monocyte-derivedmacrophages was assayed by RNase protection. Neither message was presentin freshly isolated monocytes but both messages were found at day 2 ofin vitro differentiation of the monocytes into macrophages and persistedthrough 6 days of culture. The quantity of the two messages wasapproximately equivalent throughout the differentiation period. Incontrast, similar analyes of neural tissues revealed that only fulllength message predicted to encode the full length extracellular form ofPAF-AH is expressed.

EXAMPLE 5

Genomic human plasma PAF-AH sequences were also isolated. The structureof the PAF-AH gene was determined by isolating lambda and P1 phageclones containing human genomic DNA by DNA hybridization underconditions of high stringency. Fragments of the phage clones weresubcloned and sequenced using primers designed to anneal at regularintervals throughout the cDNA clone sAH 406-3. In addition, newsequencing primers designed to anneal to the intron regions flanking theexons were used to sequence back across the exon-intron boundaries toconfirm the sequences. Exon/intron boundaries were defined as the pointswhere the genomic and cDNA sequences diverged. These analyses revealedthat the human PAF-AH gene is comprised of 12 exons.

Exons 1, 2, 3, 4, 5, 6, and part of 7 were isolated from a male fetalplacental library constructed in lamda FIX (Stratagene). Phage plaqueswere blotted onto nitrocellulose and prehybridized and hybridized in 50%formamide, 0.75M sodium chloride, 75 mM sodium citrate, 50 mM sodiumphosphate (pH 6.5), 1% polyvinyl pyrolidine, 1% Ficoll, 1% bovine serumalbumin, and 50 ng/ml sonicated salmon sperm DNA. The hybridizationprobe used to identify a phage clone containing exons 2-6 and part of 7consisted of the entire cDNA clone sAH 406-3. A clone containing exon 1was identified using a fragment derived from the 5' end of the cDNAclone (nucleotides 1 to 312 of SEQ ID NO:7). Both probes were labelledwith ³² P by hexamer random priming. After overnight hybridization at42° C., blots were washed extensively in 30 mM sodium chloride, 3 mMsodium citrate, 0.1% SDS at 42° C. The DNA sequences of exons 1, 2, 3,4, 5, and 6 along with partial surrounding intron sequences are set outin SEQ ID NOs: 9, 10, 11, 12, 13, and 14, respectively.

The remainder of exon 7 as well as exons 8, 9, 10, 11, and 12 weresubcloned from a P1 clone isolated from a human P1 genomic library. P1phage plaques were blotted onto nitrocellulose and prehybridized andhybridized in 0.75M sodium chloride, 50 mM sodium phosphate (pH 7.4), 5mM EDTA, 1% polyvinyl pyrolidine, 1% Ficoll, 1% bovine serum albumin,0.5% SDS, and 0.1 mg/ml total human DNA. The hybridization probe,labeled with ³² P by hexamer random priming, consisted of a 2.6 kb EcoRIfragment of genomic DNA derived from the 3' end of a lambda cloneisolated above. This fragment contained exon 6 and the part of exon 7present on the phage clone. After overnight hybridization at 65° C.,blots were washed as described above. The DNA sequences of exons 7, 8,9, 10, 11, and 12 along with partial surrounding intron sequences areset out in SEQ ID NOs: 15, 16, 17, 18, 19, and 20, respectively.

EXAMPLE 6

Full length plasma PAF-AH cDNA clones were isolated from mouse, canine,bovine and chicken spleen cDNA libraries and a partial rodent clone wasisolated from a rat thymus cDNA library. The clones were identified bylow stringency hybridization to the human cDNA (hybridization conditionswere the same as described for exons 1 through 6 in Example 5 aboveexcept that 20% formamide instead of 50% formamide was used). A 1 kbHindIII fragment of the human PAF-AH sAH 406-3 cDNA clone (nucleotides309 to 1322 of SEQ ID NO:7) was used as a probe. In addition, a partialmonkey clone was isolated from macaque brain cDNA by PCR using primersbased on nucleotides 285 to 303 and 851 to 867 of SEQ ID NO:7. Thenucleotide and deduced amino acid sequences of the mouse, canine,bovine, chicken, rat, and macaque cDNA clones are set out in SEQ IDNOs:21 and 31, 22 and 32, 23 and 33, 24 and 34, 25 and 35, and 26 and36, respectively.

A comparison of the deduced amino acid sequences of the cDNA clones withthe human cDNA clone results in the amino acid percentage identityvalues set out in Table 3 below.

                  TABLE 3                                                         ______________________________________                                        Human         Dog    Mouse     Bovine                                                                              Chicken                                  ______________________________________                                        Dog     80        100    64      82    50                                     Mouse   66        64     100     64    47                                     Monkey  92        82     69      80    52                                     Rat     74        69     82      69    55                                     Bovine  82        82     64      100   50                                     Chicken 50        50     47      50    100                                    ______________________________________                                    

About 38% of the residues are completely conserved in all the sequences.The most divergent regions are at the amino terminal end (containing thesignal sequence) and the carboxyl terminal end which are shown inExample 10 as not critical for enzymatic activity. TheGly-Xaa-Ser-Xaa-Gly motif (SEQ ID NO:27) found in neutral lipases andother esterases was conserved in the bovine, canine, mouse, rat andchicken PAF-AH. The central serine of this motif serves as the activesite nucleophile for these enzymes. The predicted aspartate andhistidine components of the active site (Example 10A) were alsoconserved. The human plasma PAF-AH of the invention therefore appears toutilize a catalytic triad and may assume the α/β hydrolase conformationof the neutral lipases even though it does not exhibit other sequencehomology to the lipases.

Moreover, human plasma PAF-AH is expected to have a region that mediatesits specific interaction with the low density and high densitylipoprotein particles of plasma. Interaction with these particles may bemediated by the N-terminal half of the molecule which has largestretches of amino acids highly conserved among species but does notcontain the catalytic triad of the enzyme.

EXAMPLE 7

To determine whether human plasma PAF-AH cDNA clone sAH 406-3 (Example3) encodes a protein having PAF-AH activity, the pRc/CMV expressionconstruct was transiently expressed in COS 7 cells. Three days followingtransfection by a DEAE Dextran method, COS cell media was assayed forPAF-AH activity.

Cells were seeded at a density of 300,000 cells per 60 mm tissue culturedish. The following day, the cells were incubated in DMEM containing 0.5mg/ml DEAE dextran, 0.1 mM chloroquine and 5-10 μg of plasmid DNA for 2hours. Cells were then treated with 10% DMSO in phosphate-bufferedsaline for 1 minute, washed with media and incubated in DMEM containing10% fetal calf serum previously treated with diisopropyl fluorophosphate(DFP) to inactivate endogenous bovine serum PAF-AH. After 3 days ofincubation, media from transfected cells were assayed for PAF-AHactivity. Assays were conducted in the presence and absence of either 10mM EDTA or 1 mM DFP to determine whether the recombinant enzyme wascalcium-independent and inhibited by the serine esterase inhibitor DFPas previously described for plasma PAF-AH by Stafforini et al. (1987),supra. Negative controls included cells transfected with pRc/CMV eitherlacking an insert or having the sAH 406-3 insert in reverse orientation.

PAF-AH activity in transfectant supernatants was determined by themethod of Stafforini et al. (1990), supra, with the followingmodifications. Briefly, PAF-AH activity was determined by measuring thehydrolysis of ³ H-acetate from acetyl-³ H! PAF (New England Nuclear,Boston, Mass.). The aqueous free ³ H-acetate was separated from labeledsubstrate by reversed-phase column chromatography over octadecylsilicagel cartridges (Baker Research Products, Phillipsburg, Pa.). Assays werecarried out using 10 μl transfectant supernatant in 0.1M Hepes buffer,pH 7.2, in a reaction volume of 50 μl. A total of 50 pmoles of substratewere used per reaction with a ratio of 1:5 labeled: cold PAF. Reactionswere incubated for 30 minutes at 37° C. and stopped by the addition of40 μl of 10M acetic acid. The solution was then washed through theoctadecylsilica gel cartridges which were then rinsed with 0.1M sodiumacetate. The aqueous eluate from each sample was collected and countedin a liquid scintillation counter for one minute. Enzyme activity wasexpressed in counts per minute.

As shown in FIG. 2, media from cells transfected with sAH 406-3contained PAF-AH activity at levels 4-fold greater than background. Thisactivity was unaffected by the presence of EDTA but was abolished by 1mM DFP. These observations demonstrate that clone sAH 406-3 encodes anactivity consistent with the human plasma enzyme PAF-AH.

EXAMPLE 8

Human plasma PAF-AH cDNA was expressed in E. coli and yeast and stablyexpressed in mammalian cells by recombinant methods.

A. Expression in E. coli

PCR was used to generate a protein coding fragment of human plasmaPAF-AH cDNA from clone sAH 406-3 which was readily amenable tosubcloning into an E. coli expression vector. The subcloned segmentbegan at the 5' end of the human gene with the codon that encodes Ile₄₂(SEQ ID NO:8), the N-terminal residue of the enzyme purified from humanplasma. The remainder of the gene through the native termination codonwas included in the construct. The 5' sense PCR primer utilized was:

SEQ ID NO:28 5' TATTCTAGAATTATGATACAAGTATTAATGGCTGCTGCAAG

3' and contained an XbaI cloning site as well as a translationinitiation codon (underscored). The 3' antisense primer utilized was:

SEQ ID NO:29 5' ATTGATATCCTAATTGTATTTCTCTATTCCTG 3'

and encompassed the termination codon of sAH 406-3 and contained anEcoRV cloning site. PCR reactions were performed essentially asdescribed in Example 3. The resulting PCR product was digested with XbaIand EcoRV and subcloned into a pBR322 vector containing the Trp promoterdeBoer et al., PNAS, 80:21-25 (1983)! immediately upstream of thecloning site. E. coli strain XL-1 Blue was transformed with theexpression construct and cultured in L broth containing 100 μg/ml ofcarbenicillin. Transformants from overnight cultures were pelleted andresuspended in lysis buffer containing 50 mM Tris-HCl pH 7.5, 50 mMNaCl, 10 mM CHAPS, 1 mM EDTA, 100 μg/ml lysozyme, and 0.05trypsin-inhibiting units (TIU)/ml Aprotinin. Following a 1 hourincubation on ice and sonication for 2 minutes, the lysates were assayedfor PAF-AH activity by the method described in Example 4. E. colitransformed with the expression construct (designated trp AH) generateda product with PAF-AH activity. See Table 6 in Example 9.

Constructs including three additional promoters, the tacII promoter(deBoer, supra), the arabinose (ara) B promoter from Salmonellatyphimurium Horwitz et al., Gene, 14: 309-319 (1981)!, and thebacteriophage T7 promoter, were also utilized to drive expression ofhuman PAF-AH sequences in E. coli. Constructs comprising the Trppromoter (pUC trp AH), the tacII promoter (pUC tac AH), and the araBpromoter (pUC ara AH) were assembled in plasmid pUC19 (New EnglandBiolabs, Mass.) while the construct comprising the T7 promoter (PET AH)was assembled in plasmid pET15B (Novagen, Madison, Wisc.). A constructcontaining a hybrid promoter, pHAB/PH, consisting of the araB promoterfused to the ribosome binding sites of the T7 promoter region was alsoassembled in pET15B. All E. coli constructs produced PAF-AH activitywithin a range of 20 to 50 U/ml/OD₆₀₀. This activity corresponded to atotal recombinant protein mass of >1% of the total cell protein.

Several E. coli expression constructs were also evaluated which producePAF-AH with extended amino termini. The N-terminus of natural plasmaPAF-AH was identified as Ile₄₂ by amino acid sequencing (Example 2).However, the sequence immediately upstream of Ile₄₂ does not conform toamino acids found at signal sequence cleavage sites i.e., the"-3-1-rule" is not followed, as lysine is not found at position -1; seevon Heijne, Nuc. Acids Res., 14:4683-4690 (1986)!. Presumably a moreclassical signal sequence (M₁ -A₁₇) is recognized by the cellularsecretion system, followed by endoproteolytic cleavage. The entirecoding sequence for PAF-AH beginning at the initiating methionine(nucleotides 162 to 1487 of SEQ ID NO:7) was engineered for expressionin E. coli using the trp promoter. As shown in Table 4, this constructmade active PAF-AH, but expression was at about one fiftieth of thelevel of the original construct beginning at Ile₄₂. Another expressionconstruct, beginning at Val₁₈ (nucleotides 213 to 1487 of SEQ ID NO:7),produced active PAF-AH at about one third the level of the originalconstruct. These results suggest that amino terminal end extensions arenot critical or necessary for activity of recombinant PAF-AH produced inE. coli.

                  TABLE 4                                                         ______________________________________                                                        PAF-AH activity                                                               (U/ml/OD.sub.600)                                             Construct         Lysate  Media                                               ______________________________________                                        pUC trp AH        177.7   0.030                                               pUC trp AH Met.sub.1                                                                            3.1     0.003                                               pUC trp AH Val.sub.18                                                                           54.6    0.033                                               ______________________________________                                    

Recombinant human PAF-AH was also produced in E. coli using a low copynumber plasmid and a promoter that can be induced by the addition ofarabinose to the culture. The PAF-AH protein encoded within the plasmidbegins at the methionine forty-six residues from the N-terminus of thepolypeptide encoded by full length PAF-AH cDNA.

The plasmid used for production of human PAF-AH in bacterial cells waspBAR2/PH.2, which is a pBR322-based plasmid that carries (1) nucleotides297 to 1487 of SEQ ID NO:7 encoding human PAF-AH beginning with themethionine codon at position 46, (2) the araB-C promoters and araC genefrom the arabinose operon of Salmonella typhimurium, (3) a transcriptiontermination sequence from the bacteriophage T7, and (4) a replicationorigin from bacteriophage f1.

Specifically, pBAR2/PH.2 included the following segments of DNA: (1)from the destroyed AatII site at position 1994 to the EcoRI site atnucleotide 6274, vector sequence containing an origin of replication andgenes encoding resistance to either ampicillin or tetracycline derivedfrom the bacterial plasmid pBR322; (2) from the EcoRI site at position6274 to the XbaI site at position 131, DNA from the Salmonellatyphimurium arabinose operon (Genbank accession numbers M11045, M11046,M11047, J01797); (3) from the XbaI site at position 131 to the NcoI siteat position 170, DNA containing a ribosome binding site from pET-21b(Novagen, Madison, Wis.); (4) from the NcoI site at position 170 to theXhoI site at position 1363, human PAF-AH cDNA sequence; and (5) from theXhoI site at position 1363 to the destroyed AatII site at position 1993,a DNA fragment from pET-21b (Novagen) that contains a transcriptiontermination sequence from bacteriophae T7 and an origin of replicationfrom bacteriophage f1.

Expression of PAF-AH in pBAR2/PH.2 is under the control of the araBpromoter, which is tightly repressed in the presence of glucose andabsence of arabinose, but functions as a strong promoter whenL-arabinose is added to cultures depleted of glucose. Selection forcells containing the plasmid can be accomplished through the addition ofeither ampicillin (or related antibiotics) or tetracycline to theculture medium.

The E. coli strain used for production of PAF-AH is MC1061 (ATCC 53338),which carries a deletion of the arabinose operon and thereby cannotmetabolize arabinose. The advantage of using a strain that is unable tobreak down arabinose is that the inducer (arabinose) for production ofPAF-AH is not depleted from the medium during the induction period,resulting in higher levels of PAF-AH compared to that obtained withstrains that are capable of metabolizing arabinose. MC1061 is also aleucine auxotroph and was cultivated by batch-fed process using adefined media containing casamino acids that complement the leucinemutation. Cells were grown at 30° C. in batch media containing 2 gm/Lglucose. Glucose serves the dual purpose of carbon source for cellgrowth, and repressor of the arabinose promoter. When batch glucoselevels were depleted (<50 mg/L), a nutrient feed (containing 300 gm/Lglucose) was started. The feed was increased linearly for 16 hours at arate which limited acid bi-product formation. At this point, thenutrient feed was switched to media containing glycerol instead ofglucose. Simultaneously, 500 gm/L L-arabinose was added to a finalconcentration of 5 gm/L. The glycerol feed was kept at a constant feedrate for 22 hours. Cells were harvested using hollow-fiber filtration toconcentrate the suspension approximately 10-fold. Cell paste was storedat -70° C. A final cell mass of about 80 gm/L was obtained (OD₆₀₀=50-60) with a PAF-AH activity of 65-70 U/OD/ml representing about 10%of total cell protein. The final culture volume of about 75 literscontained 50-60 gm PAF-AH.

B. Expression in Yeast Cells

Recombinant human PAF-AH was also expressed in Saccharomyces cerevisiae.The yeast ADH2 promoter was used to drive rPAF-AH expression andproduced 7 U/ml/OD₆₀₀ (Table 5 below).

                  TABLE 5                                                         ______________________________________                                                                         Enzyme Activity                              Construct Promoter  Strain       (U/ml/OD)                                    ______________________________________                                        pUC tac AH                                                                              tac       E. coli W3110                                                                              30                                           pUC trp AH                                                                              trp       E. coli W3110                                                                              40                                           pUC ara AH                                                                              araB      E. coli W3110                                                                              20                                           pET AH    T7        E. coli BL21 (DE3)                                                                         50                                                               (Novagen)                                                 pHAB/PH   araB/T7   E. coli XL-1 34                                           pBAR2/PH.2                                                                              araB      MC1061       90                                           pYep ADH2 AH                                                                            ADH2      Yeast BJ2.28  7                                           ______________________________________                                    

C. Expression of PAF-AH in mammalian cells

1. Expression of Human PAF-AH cDNA Constructs

Plasmids constructed for expression of PAF-AH, with the exception ofpSFN/PAFAH.1, employ a strong viral promoter from cytomegalovirus, apolyadenylation site from the bovine growth hormone gene, and the SV40origin of replication to permit high copy number replication of theplasmid in COS cells. Plasmids were electroporated into cells.

A first set of plasmids was constructed in which the 5' flankingsequence (pDC1/PAFAH.1) or both the 5' or 3' flanking sequences(PDC1/PAFAH.2) of the human PAF-AH cDNA were replaced with flankingsequences from other genes known to be expressed at high levels inmammalian cells. Transfection of these plasmids into COS, CHO or 293cells led to production of PAF-AH at about the same level (0.01 units/mlor 2-4 fold above background) as that cited for clone sAH 406-3 inExample 7 after transient transfection of COS cells. Another plasmid wasconstructed which included a Friend spleen focus-forming virus promoterinstead of the cytomegalovirus promoter. The human PAF-AH cDNA wasinserted into plasmid pmH-neo Hahn et al., Gene, 127: 267 (1993)! undercontrol of the Friend spleen focus-forming virus promoter. Transfectionof the myeloma cell line NSO with the plasmid which was designatedpSFN/PAFAH.1 and screening of several hundred clones resulted in theisolation of two transfectants (4B1 1 and 1C 11) that made 0.15-0.5units/ml of PAF-AH activity. Assuming a specific activity of 5000units/milligram, the productivity of these two NSO transfectantscorresponds to about 0.1 mg/liter.

2. Expression of Mouse-Human Chimeric PAF-AH Gene Constructs

A construct (pRc/MS9) containing the cDNA encoding mouse PAF-AH in themammalian expression vector pRc/CMV resulted in production of secretedPAF-AH at the level of 5-10 units/ml (1000 fold above background) aftertransfection into COS cells. Assuming that the specific activity of themouse PAF-AH is about the same as that of the human enzyme, the mousecDNA is therefore expressed at a 500-1000 fold higher level than is thehuman PAF-AH cDNA.

To examine the difference between the expression levels of human andmouse PAF-AH in COS cells, two mouse-human chimeric genes wereconstructed and tested for expression in COS cells. The first of theseconstructs, pRc/PH.MHC1, contains the coding sequence for the N-terminal97 amino acids of the mouse PAF-AH polypeptide (SEQ ID NO:21) fused tothe C-terminal 343 amino acids of human PAF-AH in the expression vectorpRc/CMV (Invitrogen, San Diego, Calif.). The second chimeric gene, inplasmid pRc/PH.MHC2, contains the coding sequence for the N-terminal 40amino acids of the mouse PAF-AH polypeptide fused to the C-terminal 400residues of human PAF-AH in pRc/CMV. Transfection of COS cells withpRc/PH.MHC1 led to accumulation of 1-2 units/ml of PAF-AH activity inthe media. Conditioned media derived from cells transfected withpRc/PH.MHC2 was found to contain only 0.01 units/ml of PAF-AH activity.From these experiments, it appears that the difference in expressionlevel between mouse and human PAF-AH genes is attributable at least inpart to the polypeptide segment between the residues 40 and 97, or thecorresponding RNA or DNA segment encoding this region of the PAF-AHprotein.

3. Recoding of the First 290 bp of the PAF-AH Coding Sequence

One hypothesis for the low level of human PAF-AH synthesized intransfected mammalian cells is that the codons utilized by the naturalgene are suboptimal for efficient expression. However, it does not seemlikely that codon usage can account for 500-1000 fold difference inexpression levels between the mouse and human genes because optimizingcodons generally has at most only a 10-fold effect on expression. Asecond hypothesis to explain the difference between the mouse and humanPAF-AH expression levels is that the human PAF-AH mRNA in the 5' codingregion forms a secondary structure that leads to either relatively rapiddegradation of the mRNA or causes inefficient translation initiation orelongation.

To test these hypotheses, a synthetic fragment encoding the authentichuman PAF-AH protein from the amino-terminus to residue 96 but in whichmost of the codons have been substituted ("recoded") with a codon of adifferent sequence but encoding the same amino acid was constructed.Changing the second codon from GTG to GTA resulted in the creation of anAsp718 site, which was at one end of the synthetic fragment and which ispresent in the mouse cDNA. The other end of the fragment contained theBamHI site normally found at codon 97 of the human gene. Theapproximately 290 bp Asp718/BamHI fragment was derived from a PCRfragment that was made using the dual asymmetric PCR approach forconstruction of synthetic genes described in Sandhu et al.,Biotechniques, 12: 14-16 (1992). The synthetic Asp718/BamHI fragment wasligated with DNA fragments encoding the remainder of the human PAF-AHmolecule beginning with nucleotide 453 of SEQ ID NO:7 such that asequence encoding authentic human PAF-AH enzyme was inserted into themammalian expression vector pRc/CMV (Invitrogen, San Diego) to createplasmid pRc/HPH.4. The complete sequence of the recoded gene is set outin SEQ ID NO:30. The 5' flanking sequence adjacent to the human PAF-AHcoding sequence in pRc/HPH.4 is from that of a mouse cDNA encodingPAF-AH in pRc/MS9 (nucleotides 1 to 116 of SEQ ID NO:21).

To test expression of human PAF-AH from pRc/HPH.4, COS cells weretransiently transfected with pRc/HPH.4 (recoded human gene), pRc/MS9(mouse PAF-AH), or pRc/PH.MHC1 (mouse-human hybrid 1). The conditionedmedia from the transfected cells were tested for PAF-AH activity andfound to contain 5.7 units/ml (mouse gene), 0.9 units/ml (mouse-humanhybrid 1), or 2.6 units/ml (recoded human gene). Thus, the strategy ofrecoding the first 290 bp of coding sequence of human PAF-AH wassuccessful in boosting expression levels of human PAF-AH from a fewnanograms/ml to about 0.5 microgram/ml in a transient COS celltransfection. The recoded PAF-AH gene from pRc/HPH.4 will be insertedinto a mammalian expression vector containing the dihydrofolatereductase (DHFR) gene and DHFR-negative chinese hamster ovary cells willbe transfected with the vector. The transfected cells will be subjectedto methotrexate selection to obtain clones making high levels of humanPAF-AH due to gene amplification.

EXAMPLE 9

Recombinant human plasma PAF-AH (beginning at Ile₄₂ expressed in E. coliwas purified to a single Coomassie-stained SDS-PAGE band by variousmethods and assayed for activities exhibited by the native PAF-AHenzyme.

A. Purification of Recombinant PAF-AH

The first purification procedure utilized is similar to that describedin Example 1 for native PAF-AH. The following steps were performed at 4°C. Pellets from 50 ml PAF-AH producing E. coli (transformed withexpression construct trp AH) were lysed as described in Example 8.Solids were removed by centrifugation at 10,000 g for 20 minutes. Thesupernatant was loaded at 0.8 ml/minute onto a Blue Sepharose Fast Flowcolumn (2.5 cm×4 cm; 20 ml bed volume) equilibrated in buffer D (25 mMTris-HCl, 10 mM CHAPS, 0.5M NaCl, pH 7.5). The column was washed with100 ml buffer D and eluted with 100 ml buffer A containing 0.5M KSCN at3.2 ml/minute. A 15 ml active fraction was loaded onto a 1 ml CuChelating Sepharose column equilibrated in buffer D. The column waswashed with 5 ml buffer D followed by elution with 5 ml of buffer Dcontaining 100 mM imidazole with gravity flow. Fractions containingPAF-AH activity were analyzed by SDS-PAGE.

The results of the purification are shown in Table 6 wherein a unitequals μmol PAF hydrolysis per hour. The purification product obtainedat 4° C. appeared on SDS-PAGE as a single intense band below the 43 kDamarker with some diffuse staining directly above and below it. Therecombinant material is significantly more pure and exhibits greaterspecific activity when compared with PAF-AH preparations from plasma asdescribed in Example 1.

                                      TABLE 6                                     __________________________________________________________________________                Total    Specific                                                         Activity                                                                          Act.     Activity                                                                          % Recovery                                                                           Fold                                          Volume  (units/                                                                           (units ×                                                                    Prot Conc                                                                          (units/                                                                           of Activity                                                                          Purification                                  Sample                                                                            (ml)                                                                              ml) 10.sup.3)                                                                         (mg/mL)                                                                            mg) Step                                                                             Cum.                                                                              Step                                                                             Cum.                                       __________________________________________________________________________    Lysate                                                                            4.5 989 4451                                                                              15.6 63  100                                                                              100 1  1                                          Blue                                                                              15  64  960 0.07 914 22 22  14.4                                                                             14.4                                       Cu  1   2128                                                                              2128                                                                              0.55 3869                                                                              220                                                                              48  4.2                                                                              61                                         __________________________________________________________________________

When the same purification protocol was performed at ambienttemperature, in addition to the band below the 43 kDa marker, a group ofbands below the 29 kDa marker correlated with PAF-AH activity of assayedgel slices. These lower molecular weight bands may be proteolyticfragments of PAF-AH that retain enzymatic activity.

A different purification procedure was also performed at ambienttemperature. Pellets (100 g) of PAF-AH-producing E. coli (transformedwith the expression construct pUC trp AH) were resuspended in 200 ml oflysis buffer (25 mM Tris, 20 mM CHAPS, 50 mM NaCl, 1 mM EDTA, 50 μg/mlbenzamidine, pH 7.5) and lysed by passing three times through amicrofluidizer at 15,000 psi. Solids were removed by centrifugation at14,300×g for 1 hour. The supernatant was diluted 10-fold in dilutionbuffer 25 mM MES (2- N-morpholino!ethanesulfonic acid), 10 mM CHAPS, 1mM EDTA, pH 4.9! and loaded at 25 ml/minute onto an S Sepharose FastFlow Column (200 ml) (a cation exchange column) equilibrated in Buffer E(25 mM MES, 10 mM CHAPS, 1 mM EDTA, 50 mM NaCl, pH 5.5). The column waswashed with 1 liter of Buffer E, eluted with 1M NaCl, and the eluate wascollected in 50 ml fractions adjusted to pH 7.5 with 0.5 ml of 2M Trisbase. Fractions containing PAF-AH activity were pooled and adjusted to0.5M NaCl. The S pool was loaded at 1 ml/minute onto a Blue SepharoseFast Flow column (2.5 cm×4 cm; 20 ml) equilibrated in Buffer F (25 mMTris, 10 mM CHAPS, 0.5M NaCl, 1mM EDTA, pH 7.5). The column was washedwith 100 ml Buffer F and eluted with 100 ml Buffer F containing 3M NaClat 4 ml/minute. The Blue Sepharose Fast Flow chromatography step wasthen repeated to reduce endotoxin levels in the sample. Fractionscontaining PAF-AH activity were pooled and dialyzed against Buffer G (25mM Tris pH 7.5, 0.5M NaCl, 0.1% Tween 80, 1 mM EDTA).

The results of the purification are shown in Table 7 wherein a unitequals μmol PAF hydrolysis per hour.

                                      TABLE 7                                     __________________________________________________________________________                Total    Specific                                                         Activity                                                                          Act.     Activity                                                                          % Recovery                                                                           Fold                                          Volume  (units/                                                                           (units ×                                                                    Prot Conc                                                                          (units/                                                                           of Activity                                                                          Purification                                  Sample                                                                            (ml)                                                                              ml) 10.sup.3)                                                                         (mg/mL)                                                                            mg) Step                                                                             Cum.                                                                              Step                                                                             Cum.                                       __________________________________________________________________________    Lysate                                                                            200 5640                                                                              1128                                                                              57.46                                                                              98  100                                                                              100 1  1                                          S   111 5742                                                                              637 3.69 1557                                                                              57 56  16 16                                         Blue                                                                              100 3944                                                                              394 0.84 4676                                                                              35 62  3  48                                         __________________________________________________________________________

The purification product obtained appeared on SDS-PAGE as a singleintense band below the 43 kDa marker with some diffuse staining directlyabove and below it. The recombinant material is significantly more pureand exhibits greater specific activity when compared with PAF-AHpreparations from plasma as described in Example 1.

Yet another purification procedure contemplated by the present inventioninvolves the following cell lysis, clarification, and first columnsteps. Cells are diluted 1:1 in lysis buffer (25 mM Tris pH 7.5, 150 mMNaCl, 1% Tween 80, 2 mM EDTA). Lysis is performed in a chilledmicrofluidizer at 15,000-20,000 psi with three passes of the material toyield >99% cell breakage. The lysate is diluted 1:20 in dilution buffer(25 mM Tris pH 8.5, 1 mM EDTA) and applied to a column packed withQ-Sepharose Big Bead chromatography media (Pharmacia) and equilibratedin 25 mM Tris pH 8.5, 1 mM EDTA, 0.015% Tween 80. The eluate is diluted1:10 in 25 mM MES pH 5.5, 1.2M Ammonium sulfate, 1 mM EDTA and appliedto Butyl Sepharose chromography media (Pharmacia) equilibrated in thesame buffer. PAF-AH activity is eluted in 25 mM MES pH. 5.5, 0.1% Tween80, 1mM EDTA.

Still another method contemplated by the invention for purifyingenzymatically-active PAF-AH from E. coli includes the steps of: (a)preparing an E. coli extract which yields solubilized PAF-AH supernatantafter lysis in a buffer containing CHAPS; (b) dilution of the saidsupernatant and application to a anion exchange column equilibrated atabout pH 8.0; (c) eluting PAF-AH enzyme from said anion exchange column;(d) applying said adjusted eluate from said anion exchange column to ablue dye ligand affinity column; (e) eluting the said blue dye ligandaffinity column using a buffer comprising 3.0M salt; (f) dilution of theblue dye eluate into a suitable buffer for performing hydroxylapatitechromatography; (g) performing hydroxylapatite chromatography wherewashing and elution is accomplished using buffers (with or withoutCHAPS); (h) diluting said hydroxylapatite eluate to an appropriate saltconcentration for cation exchange chromatography; (i) applying saiddiluted hydroxylapatite eluate to a cation exchange column at a pHranging between approximately 6.0 to 7.0; (j) elution of PAF-AH fromsaid cation exchange column with a suitable formulation buffer; (k)performing cation exchange chromatography in the cold; and (l)formulation of PAF-AH in liquid or frozen form in the absence of CHAPS.

Preferably in step (a) above the lysis buffer is 25 mM Tris, 100 mMNaCl, 1 mM EDTA, 20 mM CHAPS, pH 8.0; in step (b) the dilution of thesupernatant for anion exchange chromatography is 3-4 fold into 25 mMTris, 1 mM EDTA, 10 mM CHAPS, pH 8.0 and the column is a Q-Sepharosecolumn equilibrated with 25 mM Tris, 1 mM EDTA, 50 mM NaCl, 10 mM CHAPS,pH 8.0; in step (c) the anion exchange column is eluted using 25 mMTris, 1 mM EDTA, 350 mM NaCl, 10 mM CHAPS, pH 8.0; in step (d) theeluate from step (c) is applied directly onto a blue dye affinitycolumn; in step (e) the column is eluted with 3M NaCl, 10 mM CHAPS, 25mM Tris, pH 8.0 buffer; in step (f) dilution of the blue dye eluate forhydroxylapatite chromatography is accomplished by dilution into 10 mMsodium phosphate, 100 mM NaCl, 10 mM CHAPS, pH 6.2; in step (g)hydroxylapatite chromatography is accomplished using a hydroxylapatitecolumn equilibrated with 10 mM sodium phosphate, 100 mM NaCl, 10 mMCHAPS and elution is accomplished using 50 mM sodium phosphate, 100 mMNaCl (with or without) 10 mM CHAPS, pH 7.5; in step (h) dilution of saidhydroxylapatite eluate for cation exchange chromatography isaccomplished by dilution into a buffer ranging in pH from approximately6.0 to 7.0 comprising sodium phosphate (with or without CHAPS); in step(i) a S Sepharose column is equilibrated with 50 mM sodium phosphate,(with or without) 10 mM CHAPS, pH 6.8; in step (j) elution isaccomplished with a suitable formulation buffer such as potassiumphosphate 50 mM, 12.5 mM aspartic acid, 125 mM NaCl, pH 7.5 containing0.01% Tween-80; and in step (k) cation exchange chromatrography isaccomplished at 2°-8° C. Examples of suitable formulation buffers foruse in step (l) which stabilize PAF-AH include 50 mM potassiumphosphate, 12.5 mM Aspartic acid, 125 mM NaCl pH 7.4 (approximately,with and without the addition of Tween-80 and or Pluronic F68) or 25 mMpotassium phosphate buffer containing (at least) 125 mM NaCl, 25 mMarginine and 0.01% Tween-80 (with or without Pluronic F68 atapproximately 0.1 and 0.5%).

B. Activity of Recombinant PAF-AH

The most remarkable property of the PAF acetylhydrolase is its markedspecificity for substrates with a short residue at the sn-2 position ofthe substrate. This strict specificity distinguishes PAF acetylhydrolasefrom other forms of PLA₂. Thus, to determine if recombinant PAF-AHdegrades phospholipids with long-chain fatty acids at the sn-2 position,hydrolysis of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine(arachidonoylPC) was assayed since this is the preferred substrate for awell-characterized form of PLA₂. As predicted from previous studies withnative PAF-AH, this phospholipid was not hydrolyzed when incubated withrecombinant PAF-AH. In additional experiments, arachidonoylPC wasincluded in a standard PAF hydrolysis assay at concentrations rangingfrom 0 to 125 μM to determine whether it inhibited the hydrolysis of PAFby recombinant PAF-AH. These was no inhibition of PAF hydrolysis even atthe highest concentration of PAF-AH, which was 5-fold greater than theconcentration of PAF. Thus, recombinant PAF-AH exhibits the samesubstrate selectivity as the native enzyme; long chain substrates arenot recognized. Moreover, recombinant PAF-AH enzyme rapidly degraded anoxidized phospholipid (glutaroylPC) which had undergone oxidativecleavage of the sn-2 fatty acid. Native plasma PAF-AH has several otherproperties that distinguish it from other phospholipases includingcalcium-independence and resistance to compounds that modify sulfhydrylgroups or disrupt disulfides.

Both the native and recombinant plasma PAF-AH enzymes are sensitive toDFP, indicating that a serine comprises part of their active sites. Anunusual feature of the native plasma PAF acetylhydrolase is that it istightly associated with lipoproteins in circulation, and its catalyticefficiency is influenced by the lipoprotein environment. Whenrecombinant PAF-AH of the invention was incubated with human plasma(previously treated with DFP to abolish the endogenous enzyme activity),it associated with low and high density lipoproteins in the same manneras the native activity. This result is significant because there issubstantial evidence that modification of low density lipoproteins isessential for the cholesterol deposition observed in atheromas, and thatoxidation of lipids is an initiating factor in this process. PAF-AHprotects low density lipoproteins from modification under oxidizingconditions in vitro and may have such a role in vivo. Administration ofPAF-AH is thus indicated for the suppression of the oxidation oflipoproteins in atherosclerotic plaques as well as to resolveinflammation.

These results all confirm that the cDNA clone sAH 406-3 encodes aprotein with the activities of the the human plasma PAF acetylhydrolase.

EXAMPLE 10

Various other recombinant PAF-AH products were expressed in E. coli. Theproducts included PAF-AH analogs having single amino acid mutations andPAF-AH fragments.

A. PAF-AH Amino Acid Substitution Products

PAF-AH is a lipase because it hydrolyses the phospholipid PAF. While noobvious overall similarity exists between PAF-AH and other characterizedlipases, there are conserved residues found in comparisons ofstructurally characterized lipases. A serine has been identified as amember of the active site. The serine, along with an aspartate residueand a histidine residue, form a catalytic triad which represents theactive site of the lipase. The three residues are not adjacent in theprimary protein sequence, but structural studies have demonstrated thatthe three residues are adjacent in three dimensional space. Comparisonsof structures of mammalian lipases suggest that the aspartate residue isgenerally twenty-four amino acids C-terminal to the active site serine.In addition, the histidine is generally 109 to 111 amino acidsC-terminal to the active site serine.

By site-directed mutagenesis and PCR, individual codons of the humanPAF-AH coding sequence were modified to encode alanine residues and wereexpressed in E. coli. As shown in Table 8 below wherein, for example,the abbreviation "S108A" indicates that the serine residue at position273 was changed to an alanine, point mutations of Ser₂₇₃, ASP₂₉₆, orHiS₃₅₁, completely destroy PAF-AH activity. The distances between activesite residues is similar for PAF-AH (Ser to Asp, 23 amino acids; Ser toHis, 78 amino acids) and other lipases. These experiments demonstratethat Ser₂₇₃, Asp₂ 96, and His₃₅₁, are critical residues for activity andare therefore likely candidates for catalytic triad residues. Cysteinesare often critical for the functional integrity of proteins because oftheir capacity to form disulfide bonds. The plasma PAF-AH enzymecontains five cysteines. To determine whether any of the five iscritical for enzyme actvity, each cysteine was mutated individually to aserine and the resulting mutants were expressed in E. coli. As shownbelow in Table 8, a significant but not total loss of PAF-AH activityresulted from the conversion of either CyS₂₂₉ or Cys₂₉₁ to serine.Therefore, these cysteines appear to be necessary for full PAF-AHactivity. Other point mutations had little or no effect on PAF-AHcatalytic activity. In Table 8, "++++" represent wild type PAF-AHactivity of about 40-60 U/ml/OD₆₀₀, "+++" represents about 20-40U/ml/OD₆₀₀ activity, "++" represents about 10-20 U/ml/OD₆₀₀ activity,"+" represents 1-10 U/ml/OD₆₀₀ activity, and "-" indicates <1 U/ml/OD₆₀₀activity.

                  TABLE 8                                                         ______________________________________                                        Mutation       PAF-AH activity                                                ______________________________________                                        Wild type      ++++                                                           S108A          ++++                                                           S273A          -                                                              D286A          -                                                              D286N          ++                                                             D296A          -                                                              D304A          ++++                                                           D338A          ++++                                                           H351A          -                                                              H395A, H399A   ++++                                                           C67S           +++                                                            C229S          +                                                              C291S          +                                                              C334S          ++++                                                           C407S          +++                                                            ______________________________________                                    

B. PAF-AH Fragment Products

C-terminal deletions were prepared by digesting the 3' end of the PAF-AHcoding sequence with exonuclease III for various amounts of time andthen ligating the shortened coding sequence to plasmid DNA encoding stopcodons in all three reading frames. Ten different deletion constructswere characterized by DNA sequence analysis, protein expression, andPAF-AH activity. Removal of twenty-one to thirty C-terminal amino acidsgreatly reduced catalytic activity and removal of fifty-two residuescompletely destroyed activity. See FIG. 3.

Similar deletions were made at the amino terminal end of PAF-AH. Fusionsof PAF-AH with E. coli thioredoxin at the N-terminus were prepared tofacilitate consistent high level expression PAF-AH activity LaVallie etal., Bio/technology, 11:187-193 (1993)!. Removal of nineteen amino acidsfrom the naturally processed N-terminus (Ile₄₂) reduced activity by 99%while removal of twenty-six amino acids completely destroyed enzymaticactivity in the fusion protein. See FIG. 3. Deletion of twelve aminoacids appeared to enhance enzyme activity about four fold.

In subsequent purifications of PAF-AH from fresh human plasma by amethod similar to that described in Example 1 (Microcon 30 filter fromAmicon were utilized to concentrate Blue sepharose eluate instead of aCu column), two N-termini in addition to Ile₄₂ were identified, Ser₃₅and Lys₅₅. The heterogeneity may be the natural state of the enzyme inplasma or may occur during purification.

The purified material described above was also subject to analysis forglycosylation. Purified native PAF-AH was incubated in the presence orabsence of N-Glycanase, an enzyme that removes N-linked carbohydratesfrom glycoproteins. The treated PAF-AH samples were electrophoresedthrough a 12% SDS polyacrylamide gel then visualized by Western blottingusing rabbit polyclonal antisera. Protein not treated with N-Glycanasemigrated as a diffuse band of 45-50 kDa whereas the protein treated withthe glycanase migrated as a tight band of about 44 kDa, demonstratingthat native PAF-AH is glycosylated.

EXAMPLE 11

A preliminary analysis of expression patterns of human plasma PAF-AHmRNA in human tissues was conducted by Northern blot hybridization.

RNA was prepared from human cerebral cortex, heart, kidney, placenta,thymus and tonsil using RNA Stat 60 (Tel-Test "B", Friendswood, Tex.).Additionally, RNA was prepared from the human hematopoieticprecursor-like cell line, THP-1 (ATCC TIB 202), which was induced todifferentiate to a macrophage-like phenotype using the phorbol esterphorbolmyristylacetate (PMA). Tissue RNA and RNA prepared from thepremyelocytic THP-1 cell line prior to and 1 to 3 days after inductionwere electrophoresed through a 1.2% agarose formaldehyde gel andsubsequently transferred to a nitrocellulose membrane. The full lengthhuman plasma PAF-AH cDNA, sAH 406-3, was labelled by random priming andhybridized to the membrane under conditions identical to those describedin Example 3 for library screening. Initial results indicate that thePAF-AH probe hybridized to a 1.8 kb band in the thymus, tonsil, and to alesser extent, the placental RNA.

PAF is synthesized in the brain under normal physiological as well aspathophysiological conditions. Given the known pro-inflammatory andpotential neurotoxic properties of the molecule, a mechanism forlocalization of PAF synthesis or for its rapid catabolism would beexpected to be critical for the health of neural tissue. The presence ofPAF acetylhydrolase in neural tissues is consistent with it playing sucha protective role. Interestingly, both a bovine heterotrimericintracellular PAF-AH the cloning of which is described in Hattori etal., J. Biol. Chem., 269(37): 23150-23155 (1994)! and PAF-AH of theinvention have been identified in the brain. To determine whether thetwo enzymes are expressed in similar or different compartments of thebrain, the human homologue of the bovine brain intracellular PAF-AH cDNAwas cloned, and its mRNA expression pattern in the brain was compared byNorthern blotting to the mRNA expression pattern of the PAF-AH of theinvention by essentially the same methods as described in the foregoingparagraph. The regions of the brain examined by Northern blotting werethe cerebellum, medulla, spinal cord, putamen, amygdala, caudatenucleus, thalamus, and the occipital pole, frontal lobe and temporallobe of the cerebral cortex. Message of both enzymes was detected ineach of these tissues although the heterotrimeric intracellular formappeared in greater abundance than the secreted form. Northern blotanalysis of additional tissues further revealed that the heterotrimericintracellular form is expressed in a broad variety of tissues and cells,including thymus, prostate, testis, ovary, small intestine, colon,peripheral blood leukocytes, macrophages, brain, liver, skeletal muscle,kidney, pancreas and adrenal gland. This ubiquitous expression suggeststhat the heterotrimeric intracellular PAF-AH has a general housekeepingfunction within cells.

The expression of PAF-AH RNA in monocytes isolated from human blood andduring their spontaneous differentiation into macrophages in culture wasalso examined. Little or no RNA was detected in fresh monocytes, butexpression was induced and maintained during differentiation intomacrophages. There was a concomitant accumulation of PAF-AH activity inthe culture medium of the differentiating cells. Expression of the humanplasma PAF-AH transcript was also observed in the THP-1 cell RNA at 1day but not 3 days following induction. THP-1 cells did not express mRNAfor PAF-AH in the basal state.

EXAMPLE 12

PAF-AH expression in human and mouse tissues was examined by in situhybridization.

Human tissues were obtained from National Disease Research Interchangeand the Cooperative Human Tissue Network. Normal mouse brain and spinalcord, and EAE stage 3 mouse spinal cords were harvested from S/JLJ mice.Normal S/JLJ mouse embryos were harvested from eleven to eighteen daysafter fertilization.

The tissue sections were placed in Tissue Tek II cryomolds (MilesLaboratories, Inc., Naperville, Ill.) with a small amount of OCTcompound (Miles, Inc., Elkhart, Ind.). They were centered in thecryomold, the cryomold filled with OCT compound, then placed in acontainer with 2-methylbutane C₂ H₅ CH(CH₃)₂, Aldrich Chemical Company,Inc., Milwaukee, Wisc.! and the container placed in liquid nitrogen.Once the tissue and OCT compound in the cryomold were frozen, the blockswere stored at -80° C. until sectioning. The tissue blocks weresectioned at 6 μm thickness and adhered to Vectabond (VectorLaboratories, Inc., Burlingame, Calif.) coated slides and stored at -70°C. and placed at 50° C. for approximately 5 minutes to warm them andremove condensation and were then fixed in 4% paraformaldehyde for 20minutes at 4° C., dehydrated (70%, 95%, 100% ethanol) for 1 minute at 4°C. in each grade, then allowed to air dry for 30 minutes at roomtemperature. Sections were denatured for 2 minutes at 70° C. in 70%formamide/2× SSC, rinsed twice in 2× SSC, dehydrated and then air driedfor 30 minutes. The tissues were hybridized in situ with radiolabeledsingle-stranded mRNA generated from DNA derived from an internal 1 KbHindIII fragment of the PAF-AH gene (nucleotides 308 to 1323 of SEQ IDNO:7) by in vitro RNA transcription incorporation ³⁵ S-UTP (Amersham) orfrom DNA derived from the heterotrimeric intracellular PAF-AH cDNAidentified by Hattori et al. The probes were used at varying lengthsfrom 250-500 bp. Hybridization was carried out overnight (12-16 hours)at 50° C.; the ³⁵ S-labeled riboprobes (6×10⁵ cpm/section), tRNA (0.5μg/section) and diethylpyrocarbonate (depc)-treated water were added tohybridization buffer to bring it a final concentration of 50% formamide,0.3M NaCl, 20 mM Tris pH 7.5, 10% dextran sulfate, 1×Denhardt'ssolution, 100 mM dithiothretol (DTT) and 5 mM EDTA. After hybridization,sections were washed for 1 hour at room temperature in 4× SSC/10 mM DTT,then for 40 minutes at 60° C. in 50% formamide/1× SSC/10 mM DTT, 30minutes at room temperature in 2× SSC, and 30 minutes at roomtemperature in 0.1× SSC. The sections were dehydrated, air dried for 2hours, coated with Kodak NTB2 photographic emulsion, air dried for 2hours, developed (after storage at 4° C. in complete darkness) andcounterstained with hematoxylin/eosin.

A. Brain

Cerebellum. In both the mouse and the human brains, strong signal wasseen in the Purkinje cell layer of the cerebellum, in basket cells, andindividual neuronal cell bodies in the dentate nucleus (one of the fourdeep nuclei in the cerebellum). Message for the intracellular PAF-AH wasalso observed in these cell types. Additionally, plasma PAF-AH signalwas seen on individual cells in the granular and molecular layers of thegrey matter.

Hippocampus. In the human hippocampus section, individual cellsthroughout the section, which appear to be neuronal cell bodies, showedstrong signal. These were identified as polymorphic cell bodies andgranule cells. Message for the heterotrimeric intracellular PAF-AH wasalso observed in hippocampus.

Brain stem. On both human and mouse brain stem sections, there wasstrong signal on individual cells in the grey matter.

Cortex. On human cortex sections taken from the cerebral, occipital, andtemporal cortexes, and on mouse whole brain sections, individual cellsthroughout the cortex showed strong signal. These cells were identifiedas pyramidal, stellate and polymorphic cell bodies. There does notappear to be differentiation in the expression pattern in the differentlayers of the cortex. These in situ hybridization results are differentfrom the results for cerebral cortex obtained by Northern blotting. Thedifference is likely to result from the greater sensitivity of in situhybridization compared to that of Northern blotting. As in thecerebellum and hippocampus, a similar pattern of expression of theheterotrimeric intracellular PAF-AH was observed.

Pituitary. Somewhat weak signal was seen on scattered individual cellsin the pars distalis of the human tissue section.

B. Human colon

Both normal and Crohn's disease colons displayed signal in the lymphaticaggregations present in the mucosa of the sections, with the level ofsignal being slightly higher in the section from the Crohn's diseasepatient. The Crohn's disease colon also had strong signal in the laminapropria. Similarly, a high level of signal was observed in a diseasedappendix section while the normal appendix exhibited a lower but stilldetectable signal. The sections from the ulcerative colitis patientshowed no evident signal in either the lymphatic aggregations or thelamina propria.

C. Human tonsil and thymus

Strong signal was seen on scattered groups of individual cells withinthe germinal centers of the tonsil and within the thymus.

D. Human lymph node

Strong signal was observed on the lymph node section taken from a normaldonor, while somewhat weak signal was observed in the lymph nodules ofthe section from a donor with septic shock.

E. Human small intestine

Both normal and Crohn's disease small intestine had weak signal in thePeyer's patches and lamina propria in the sections, with the signal onthe diseased tissue slightly higher.

F. Human spleen and lung

Signal was not observed on any of the spleen (normal and splenic abcesssections) or lung (normal and emphysema sections) tissues.

G. Mouse spinal cord

In both the normal and EAE stage 3 spinal cords, there was strong signalin the grey matter of the spinal cord, with the expression beingslightly higher in the EAE stage 3 spinal cord. In the EAE stage 3spinal cord, cells in the white matter and perivascular cuffs, probablyinfiltrating macrophages and/or other leukocytes, showed signal whichwas absent in the normal spinal cord.

F. Mouse embryos

In the day 11 embryo signal was apparent in the central nervous systemin the fourth ventricle, which remained constant throughout the embryotime course as it developed into the cerebellum and brain stem. As theembryos matured, signal became apparent in central nervous system in thespinal cord (day 12), primary cortex and ganglion Gasseri (day 14), andhypophysis (day 16). Signal was observed in the peripheral nervoussystem (beginning on day 14 or 15) on nerves leaving the spinal cord,and, on day 17, strong signal appeared around the whiskers of theembryo. Expression was also seen in the liver and lung at day 14, thegut (beginning on day 15), and in the posterior portion of themouth/throat (beginning on day 16). By day 18, the expression patternhad differentiated into signal in the cortex, hindbrain (cerebellum andbrain stem), nerves leaving the lumbar region of the spinal cord, theposterior portion of the mouth/throat, the liver, the kidney, andpossible weak signal in the lung and gut.

G. Summary

PAF-AH mRNA expression in the tonsil, thymus, lymph node, Peyer'spatches, appendix, and colon lymphatic aggregates is consistent with theconclusions that the probable predominant in vivo source of PAF-AH isthe macrophage because these tisues all are populated with tissuemacrophages that serve as phagocytic and antigen-processing cells.

Expression of PAF-AH in inflamed tissues would be consistent with thehypothesis that a role of monocyte-derived macrophages is to resolveinflammation. PAF-AH would be expected to inactivate PAF and thepro-inflammatory phospholipids, thus down-regulating the inflammatorycascade of events initiated by these mediators.

PAF has been detected in whole brain tissue and is secreted by ratcerebellar granule cells in culture. In vitro and in vivo experimentshave demonstrated that PAF binds a specific receptor in neural tissuesand induces functional and phenotypic changes such as calciummobilization, upregulation of transcription activating genes, anddifferentiation of the neural precursor cell line, PC12. Theseobservations suggested a physiologic role for PAF in the brain, andconsistent with this, recent experiments using hippocampal tissuesection cultures and PAF analogs and antagonists have implicated PAF asan important retrograde messenger in hippocampal long term potentiation.Therefore, in addition to its pathological effect in inflammation, PAFappears to participate in routine neuronal signalling processes.Expression of the extracellular PAF-AH in the brain may serve toregulate the duration and magnitude of PAF-mediated signalling.

EXAMPLE 13

Monoclonal antibodies specific for recombinant human plasma PAF-AH weregenerated using E. coli produced PAF-AH as an immunogen.

Mouse #1342 was injected on day 0, day 19, and day 40 with recombinantPAF-AH. For the prefusion boost, the mouse was injected with theimmunogen in PBS, four days later the mouse was sacrificed and itsspleen removed sterilely and placed in 10 ml serum free RPMI 1640. Asingle-cell suspension was formed by grinding the spleen between thefrosted ends of two glass microscope slides submerged in serum free RPMI1640, supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100units/ml penicillin, and 100 μg/ml streptomycin (RPMI) (Gibco, Canada).The cell suspension was filtered through sterile 70-mesh Nitex cellstrainer (Becton Dickinson, Parsippany, N.J.), and washed twice bycentrifuging at 200 g for 5 minutes and resuspending the pellet in 20 mlserum free RPMI. Thymocytes taken from 3 naive Balb/c mice were preparedin a similar manner. NS-1 myeloma cells, kept in log phase in RPMI with11% fetal bovine serum (FBS) (Hyclone Laboratories, Inc., Logan, Utah)for three days prior to fusion, were centrifuged at 200 g for 5 minutes,and the pellet was washed twice as described in the foregoing paragraph.

One×10⁸ spleen cells were combined with 2.0×10⁷ NS-1 cells, centrifugedand the supernatant was aspirated. The cell pellet was dislodged bytapping the tube and 1 ml of 37° C. PEG 1500 (50% in 75 mM Hepes, pH8.0) (Boehringer Mannheim) was added with stirring over the course of 1minute, followed by adding 7 ml of serum free RPMI over 7 minutes. Anadditional 8 ml RPMI was added and the cells were centrifuged at 200 gfor 10 minutes. After discarding the supernatant, the pellet wasresuspended in 200 ml RPMI containing 15% FBS, 100 μM sodiumhypoxanthine, 0.4 μM aminopterin, 16 μM thymidine (HAT) (Gibco), 25units/ml IL-6 (Boehringer Mannheim) and 1.5×10⁶ thymocytes/ml and platedinto 10 Corning flat bottom 96 well tissue culture plates (Corning,Corning N.Y.).

On days 2, 4, and 6, after the fusion, 100 μl of medium was removed fromthe wells of the fusion plates and replaced with fresh medium. On day 8,the fusion was screened by ELISA, testing for the presence of mouse IgGbinding to recombinant PAF-AH. Immulon 4 plates (Dynatech, Cambridge,Mass.) were coated for 2 hours at 37° C. with 100 ng/well recombinantPAF-AH diluted in 25 mM TRIS, pH 7.5. The coating solution was aspiratedand 200ul/well of blocking solution 0.5% fish skin gelatin (Sigma)diluted in CMF-PBS! was added and incubated for 30 minutes at 37° C.Plates were washed three times with PBS with 0.05% Tween 20 (PBST) and50 μl culture supernatant was added. After incubation at 37° C. for 30minutes, and washing as above, 50 μl of horseradish peroxidaseconjugated goat anti-mouse IgG(fc) (Jackson ImmunoResearch, West Grove,Pa.) diluted 1:3500 in PBST was added. Plates were incubated as above,washed four times with PBST and 100 μL substrate, consisting of 1 mg/mlo-phenylene diamine (Sigma) and 0.1 μl/ml 30% H₂ O₂ in 100 mM Citrate,pH 4.5, was added. The color reaction was stopped in 5 minutes with theaddition of 50 μl of 15% H₂ SO₄. A₄₉₀ was read onn a plate reader(Dynatech).

Selected fusion wells were cloned twice by dilution into 96 well platesand visually scoring the number of colonies/well after 5 days.Hybridomas cloned were 90D1E, 90E3A, 90E6C, 90G11D (ATCC HB 11724), and90F2D (ATCC HB 11725).

The monoclonal antibodies produced by hybridomas were isotyped using theIsostrip system (Boehringer Mannheim, Indianapolis, Ind.). Resultsshowed that the monoclonal antibodies produced by hybridomas from fusion90 were all IgG₁.

All of the monoclonal antibodies produced by hybridomas from fusion 90functioned well in ELISA assays but were unable to bind PAF-AH onWestern blots. To generate antibodies that could recognize PAF-AH byWestern, mouse #1958 was immunized with recombinant enzyme. Hybridomaswere generated as described for fusion 90 but were screened by Westernblotting rather than ELISA to identify Western-competent clones.

For Western analyses, recombinant PAF-AH was mixed with an equal volumeof sample buffer containing 125 mM Tris, pH 6.8, 4% SDS, 100 mMdithiothreitol and 0.05% bromphenol blue and boiled for five minutesprior to loading onto a 12% SDS polyacrylamide gel (Novex). Followingelectrophoresis at 40 mAmps, proteins were electrotransferred onto apolyvinylidene fluoride membrane (Pierce) for 1 hour at 125 V in 192 mMglycine, 25 mM Tris base, 20% methanol, and 0.01% SDS. The membrane wasincubated in 20 mM Tris, 100 mM NaCl (TBS) containing 5% bovine serumalbumin (BSA, Sigma) overnight at 4° C. The blot was incubated 1 hour atroom temperature with rabbit polyclonal antisera diluted 1/8000 in TBScontaining 5% BSA, and then washed with TBS and incubated with alkalinephosphatase-conjugated goat anti-mouse IgG in TBS containing 5% BSA for1 hour at room temperature. The blot was again washed with TBS thenincubated with 0.02% 5-bromo-4-chloro-3-indolyl phosphate and 0.03%nitroblue tetrazolium in 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, and 5 mMMgCl₂. The reaction was stopped with repeated water rinses.

Selected fusion wells, the supernatants of which were positive inWestern analyses, were processed as described above. Hybridoma 143Areacted with PAF-AH in Western blots and was cloned (ATCC HB 11900).

Polyclonal antisera specific for human plasma PAF-AH was raised inrabbits by three monthly immunizations with 100 μg of purifiedrecombinant enzyme in Fruend's adjuvant.

EXAMPLE 14

Experimental studies were performed to evaluate the in vivo therapeuticeffects of recombinant PAF-AH of the invention on acute inflammationusing a rat foot edema model Henriques et al., Br. J. Pharmacol., 106:579-582 (1992)!. The results of these studies demonstrated that PAF-AHblocks PAF-induced edema. Parallel studies were done to compare theeffectiveness of PAF-AH with two commercially available PAF antagonists.

A. Preparation of PAF-AH

E. coli transformed with the PAF-AH expression vector puc trp AH werelysed in a microfluidizer, solids were centrifuged out and the cellsupernatants were loaded onto a S-Sepharose column (Pharmacia). Thecolumn was washed extensively with buffer consisting of 50 mM NaCl, 10mM CHAPS, 25 mM MES and 1 mM EDTA, pH 5.5. PAF-AH was eluted byincreasing the NaCl concentration of the buffer to 1M. Affinitychromatography using a Blue Sepharose column (Pharmacia) was then usedas an additional purification step. Prior to loading the PAF-AHpreparation on the Blue Sepharose column, the sample was diluted 1:2 toreduce the NaCl concentration to 0.5M and the pH was adjusted to 7.5.After washing the Blue Sepharose column extensively with bufferconsisting of 0.5M NaCl, 25 mM tris, 10 mM CHAPS and 1 mM EDTA, pH 7.5the PAF-AH was eluted by increasing the NaCl concentration to 3.0M.

Purity of PAF-AH isolated in this manner was generally 95% as assessedby SDS-PAGE with activity in the range of 5000-10,000 U/ml. Additionalquality controls done on each PAF-AH preparation included determiningendotoxin levels and hemolysis activity on freshly obtained raterythrocytes. A buffer containing 25 mM Tris, 10 mM CHAPS, 0.5M NaCl, pH7.5 functioned as storage media of the enzyme as well as carrier foradministration. Dosages used in experiments were based on enzymeactivity assays conducted immediately prior to experiments.

B. Induction of Edema

Six to eight-week-old female Long Evans rats (Charles River, Wilmington,Mass.), weighing 180-200 grams, were used for all experiments. Prior toexperimental manipulations, animals were anesthetized with a mixture ofthe anesthetics Ketaset (Fort Dodge Laboratories, Fort Dodge, Iowa),Rompun (Miles, Shawnee Mission, Kans.), and Ace Promazine (Aveco, FortDodge, Iowa) administered subcutaneously at approximately 2.5 mgKetaset, 1.6 mg Rompun, 0.2 mg Ace Promazine per animal per dose. Edemawas induced in the foot by administration of either PAF or zymosan asfollows. PAF (Sigma #P-1402) was freshly prepared for each experimentfrom a 19.1 mM stock solution stored in chloroform/methanol (9:1) at-20° C. Required volumes were dried down under N₂, diluted 1:1000 in abuffer containing 150 mM NaCl, 10 mM Tris pH 7.5, and 0.25% BSA, andsonicated for five minutes. Animals received 50 μl PAF (final dose of0.96 nmoles) subcutaneously between the hind foot pads, and edema wasassessed after 1 hour and again after 2 hours in some experiments.Zymosan A (Sigma #A-8800) was freshly prepared for each experiment as asuspension of 10 mg/ml in PBS. Animals received 50 μl of zymosan (finaldose of 500 μg) subcutaneously between the hind foot pads and edema wasassessed after 2 hours.

Edema was quantitated by measuring the foot volume immediately prior toadministration of PAF or zymosan and at indicated time pointpost-challenge with PAF or zymosan. Edema is expressed as the increasein foot volume in milliliters. Volume displacement measurements weremade on anesthetized animals using a plethysmometer (UGO Basile, model#7150) which measures the displaced water volume of the immersed foot.In order to insure that foot immersion was comparable from one timepoint to the next, the hind feet were marked in indelible ink where thehairline meets the heel. Repeated measurements of the same foot usingthis technique indicate the precision to be within 5%.

C. PAF-AH Administration Routes and Dosages

PAF-AH was injected locally between the foot pads, or systematically byIV injection in the tail vein. For local administration rats received100 μl PAF-AH (4000-6000 U/ml) delivered subcutaneously between theright hind foot pads. Left feet served as controls by administration of100 μl carrier (buffered salt solution). For systemic administration ofPAF-AH, rats received the indicated units of PAF-AH in 300 μl of carrieradministered IV in the tail vein. Controls received the appropriatevolume of carrier IV in the tail vein.

D. Local Administration of PAF-AH

Rats (N=4) were injected with 100 μl of PAF-AH (4000-6000 U/ml)subcutaneously between the right foot pads. Left feet were injected with100 μl carrier (buffered salt solution). Four other rats were injectedonly with carrier. All rats were immediately challenged with PAF viasubcutaneous foot injection and foot volumes assessed 1 hourpost-challenge. FIG. 4, wherein edema is expressed as average increasein foot volume (ml)±SEM for each treatment group, illustrates thatPAF-induced foot edema is blocked by local administration of PAF-AH. Thegroup which received local PAF-AH treatment prior to PAF challengeshowed reduced inflammation compared to the control injected group. Anincrease in foot volume of 0.08 ml±0.08 (SEM) was seen in the PAF-AHgroup as compared to 0.63±0.14 (SEM) for the carrier treated controls.The increase in foot volume was a direct result of PAF injection asanimals injected in the foot only with carrier did not exhibit anincrease in foot volume.

E. Intravenous Administration of PAF-AH

Rats (N=4 per group) were pretreated IV with either PAF-AH (2000 U in300 μl carrier) or carrier alone, 15 minutes prior to PAF challenge.Edema was assessed 1 and 2 hours after PAF challenge. FIG. 5, whereinedema is expressed as average increase in volume (ml)±SEM for eachtreatment group, illustrates that IV administration of PAF-AH blockedPAF induced foot edema at one and two hours post challenge. The groupwhich received 2000 U of PAF-AH given by the IV route showed a reductionin inflammation over the two hour time course. Mean volume increase forthe PAF-AH treated group at two hours was 0.10 ml±0.08 (SEM), versus0.56 ml±0.11 for carrier treated controls.

F. Comparison of PAF-AH Protection in Edema Induced by PAF or Zymosan

Rats (N=4 per group) were pretreated IV with either PAF-AH (2000 U in300 μl carrier) or carrier alone. Fifteen minutes after pretreatment,groups received either PAF or zymosan A, and foot volume was assessedafter 1 and 2 hours, respectively. As shown in FIG. 6, wherein edema isexpressed as average increase in volume (ml)±SEM for each treatmentgroup, systemic administration of PAF-AH (2000 U) was effective inreducing PAF-induced foot edema, but failed to block zymosan inducededema. A mean increase in volume of 0.08±0.02 was seen in the PAF-AHtreated group versus 0.49±0.03 for the control group.

G. Effective Dose Titration of PAF-AH Protection

In two separate experiments, groups of rats (N=3 to 4 per group) werepretreated IV with either serial dilutions of PAF-AH or carrier controlin a 300 μl volume, 15 minutes prior to PAF challenge. Both feet werechallenged with PAF (as described above) and edema was assessed after 1hour. FIG. 7 wherein edema is expressed as average increase in volume(ml)±SEM for each treatment group, illustrates the increase inprotection from PAF-induced edema in rats injected with increasingdosages of PAF-AH. In the experiments, the ID₅₀ of PAF-AH given by theIV route was found to be between 40 and 80 U per rat.

H. In Vivo Efficacy of PAF-AH as a Function of Time After Administration

In two separate experiments, two groups of rats (N=3 to 4 per group)were pretreated IV with either PAF-AH (2000 U in 300 μl carrier) orcarrier alone. After administration, groups received PAF at time pointsranging from 15 minutes to 47 hours post PAF-AH administration. Edemawas then assessed 1 hour after PAF challenge. As shown in FIG. 8,wherein edema is expressed as average increase in volume (ml)±SEM foreach treatment group, administration of 2000 U of PAF-AH protects ratsfrom PAF induced edema for at least 24 hours.

I. Pharmacokinetics of PAF-AH

Four rats received 2000 U of PAF-AH by IV injection in a 300 μl volume.Plasma was collected at various time points and stored at 4° C. andplasma concentrations of PAF-AH were determined by ELISA using a doublemAb capture assay. In brief, monoclonal antibody 90G11D (Example 13) wasdiluted in 50 mM carbonate buffer pH 9.6 at 100 ng/ml and immobilized onImmulon 4 ELISA plates overnight at 4° C. After extensive washing withPBS containing 0.05% Tween 20, the plates were blocked for 1 hour atroom temperature with 0.5% fish skin gelatin (Sigma) diluted in PBS.Serum samples diluted in PBS with 15 mM CHAPS were added in duplicate tothe washed ELISA plate and incubated for 1 hour at room temperature.After washing, a biotin conjugate of monoclonal antibody 90F2D (Example13) was added to the wells at a concentration of 5 μg/ml diluted in PBSand incubated for 1 hour at room temperature. After washing, 50 μl of a1:1000 dilution of ExtraAvidin (Sigma) was added to the wells andincubated for 1 hour at room temperature. After washing, wells weredeveloped using OPD as a substrate and quantitated. Enzyme activity wasthen calculated from a standard curve. FIG. 9, wherein data pointsrepresent means±SEM, shows that at one hour plasma enzyme levelsapproached the predicted concentration based on a 5-6 ml plasma volumefor 180-200 gram rats, mean=374 U/ml±58.2. Beyond one hour plasma levelssteadily declined, reaching a mean plasma concentration of 19.3 U/ml±3.4at 24 hours, which is still considerably higher than endogenous ratPAF-AH levels which have been found to be approximately 4 U/ml byenzymatic assays.

J. Effectiveness of PAF-AH Versus PAF Antagonists

Groups of rats (N=4 per group) were pretreated with one of threepotential antiinflammatories: the PAF antagonist CV3988 (Biomol #L-103)administered IP (2 mg in 200 μl EtOH), the PAF antagonist Alprazolam(Sigma #A-8800) administered IP (2 mg in 200 μl EtOH), or PAF-AH (2000U) administered IV. Control rats were injected IV with a 300 μl volumeof carrier. The PAF antagonists were administered IP because they aresolubilized in ethanol. Rats injected with either CV3988 or Alprazolamwere challenged with PAF 30 minutes after administration of the PAFantagonist to allow the PAF antagonist to enter circulation, whilePAF-AH and carrier-treated rats were challenged 15 minutes after enzymeadministration. Rats injected with PAF-AH exhibited a reduction inPAF-induced edema beyond that afforded by the established PAFantagonists CV3988 and Alprazolam. See FIG. 10 wherein edema isexpressed as average increase in volume (ml)±SEM for each treatmentgroup.

In summary, PAF-AH is effective in blocking edema mediated by PAF invivo. Administration of PAF-AH can be either local or systemic by IVinjection. In dosing studies, IV injections in the range of 160-2000U/rat were found to dramatically reduce PAF mediated inflammation, whilethe ID₅₀ dosage appears to be in the range of 40-80 U/rat. Calculationsbased on the plasma volume for 180-200 gram rats predicts that a plasmaconcentration in the range of 25-40 U/ml should block PAF-elicitededema. These predictions are supported by preliminary pharmacokineticstudies. A dosage of 2000 U of PAF-AH was found to be effective inblocking PAF mediated edema for at least 24 hours. At 24 hours followingadministration of PAF-AH plasma concentrations of the enzyme were foundto be approximately 25 U/ml. PAF-AH was found to block PAF-induced edemamore effectively than the two known PAF antagonists tested.

Collectively, these results demonstrate that PAF-AH effectively blocksPAF induced inflammation and may be of therapeutic value in diseaseswhere PAF is the primary mediator.

EXAMPLE 15

Recombinant PAF-AH of the invention was tested in a second in vivomodel, PAF-induced pleurisy. PAF has previously been shown to inducevascular leakage when introduced into the pleural space Henriques etal., supra!. Female rats (Charles River, 180-200 g) were injected in thetail vein with 200 μl of 1% Evans blue dye in 0.9% with 300 μlrecombinant PAF-AH (1500 μmol/ml/hour, prepared as described in Example14) or with an equivalent volume of control buffer. Fifteen minuteslater the rats received an 100 μl injection of PAF (2.0 nmol) into thepleural space. One hour following PAF challenge, rats were sacrificedand the pleural fluid was collected by rinsing the cavity with 3 mlheparinized phosphate buffered saline. The degree of vascular leak wasdetermined by the quantity of Evans blue dye in the pleural space whichwas quantitated by absorbance at 620 nm. Rats pretreated with PAF-AHwere found to have much less vascular leakage than control animals(representing more than an 80% reduction in inflammation).

The foregoing results support the treatment of subjects suffering frompleurisy with recombinant PAF-AH enzyme of the invention.

EXAMPLE 16

Recombinant PAF-AH enzyme of the invention was also tested for efficacyin a model of antigen-induced eosinophil recruitment. The accumulationof eosinophils in the airway is a characteristic feature of late phaseimmune responses which occur in asthma, rhinitis and eczema. BALB/c mice(Charles River) were sensitized by two intraperitoneal injectionsconsisting of 1 μg of ovalbumin (OVA) in 4 mg of aluminum hydroxide(Imject alum, Pierce Laboratories, Rockford, Ill.) given at a 2 weekinterval. Fourteen days following the second immunization, thesensitized mice were challenged with either aerosolized OVA or saline asa control.

Prior to challenge mice were randomly placed into four groups, with fourmice/group. Mice in groups 1 and 3 were pretreated with 140 μl ofcontrol buffer consisting of 25 mM tris, 0.5M NaCl, 1 mM EDTA and 0.1%Tween 80 given by intravenous injection. Mice in groups 2 and 4 werepretreated with 750 units of PAF-AH (activity of 5,500 units/ml given in140 μl of PAF-AH buffer). Thirty minutes following administration ofPAF-AH or buffer, mice in groups 1 and 2 were exposed to aerosolized PBSas described below, while mice in groups 3 and 4 were exposed toaerosolized OVA. Twenty-four hours later mice were treated a second timewith either 140 μl of buffer (groups 1 and 3) or 750 units of PAF-AH in140 μl of buffer (groups 2 and 4) given by intravenous injection.

Eosinophil infiltration of the trachea was induced in the sensitizedmice by exposing the animals to aerosolized OVA. Sensitized mice wereplaced in 50 ml conical centrifuge tubes (Corning) and forced to breathaerosolized OVA (50 mg/ml) dissolved in 0.9% saline for 20 minutes usinga nebulizer (Model 646, DeVilbiss Corp., Somerset, Pa.). Control micewere treated in a similar manner with the exception that 0.9% saline wasused in the nebulizer. Forty-eight hours following the exposure toaerosolized OVA or saline, mice were sacrificed and the tracheas wereexcised. Tracheas from each group were inbeded in OCT and stored at -70°until sections were cut.

To evaluate eosinophil infiltration of the trachea, tissue sections fromthe four groups of mice were stained with either Luna solution andhematoxylin-eosin solution or with peroxidase. Twelve 6 μm thicksections were cut from each group of mice and numbered accordingly. Oddnumbered sections were stained with Luna stain as follows. Sections werefixed in formal-alcohol for 5 minutes at room temperature, rinsed acrossthree changes of tap water for 2 minutes at room temperature then rinsedin two changed of dH₂ O for 1 minute at room temperature. Tissuesections were stained with Luna stain 5 minutes at room temperature(Luna stain consisting of 90 ml Weigert's Iron hematoxylin and 10 ml of1% Biebrich Scarlet). Stained slides were dipped in 1% acid alcohol sixtimes, rinsed in tap water for 1 minute at room temperature, dipped in0.5% lithium carbonate solution five times and rinsed in running tapwater for 2 minutes at room temperature. Slides were dehydrated across70%-95%-100% ethanol 1 minute each, at room temperature, then cleared intwo changes of xylene for 1 minute at room temperature and mounted inCytoseal 60.

For the peroxidase stain, even numbered sections were fixed in 4° C.acetone for 10 minutes and allowed to air dry. Two hundred μl of DABsolution was added to each section and allowed to sit 5 minutes at roomtemperature. Slides were rinsed in tap water for 5 minutes at roomtemperature and 2 drops of 1% osmic acid was applied to each section for3-5 seconds. Slides were rinsed in tap water for 5 minutes at roomtemperature and counterstained with Mayers hematoxylin at 25° C. at roomtemperature. Slides were then rinsed in running tap water for 5 minutesand dehydrated across 70%-95%-100% ethanol 1 minute each at roomtemperature. Slides were cleared through two changes of xylene for 1minute each at room temperature and mounted in Cytoseal 60.

The number of eosinophils in the submucosal tissue of the trachea wasevaluated. Trachea from mice from groups 1 and 2 were found to have veryfew eosinophils scattered throughout the submucosal tissue. As expectedtracheas from mice in group 3, which were pretreated with buffer andexposed to nebulized OVA, were found to have large numbers ofeosinophils throughout the submucosal tissue. In contrast, the tracheasfrom mice in group 4, which were pretreated with PAF-AH and exposed tonebulized OVA were found to have very few eosinophils in the submucosaltissue comparable to what was seen in the two control groups, groups 1and 2.

Thus, therapeutic treatment with PAF-AH of subjects exhibiting a latephase immune response involving the accumulation of eosinophils in theairway, such as that which occurs in asthma and rhinitis is indicated.

EXAMPLE 17

PAF-AH of the invention was also tested in a rat model for treatment ofnecrotizing enterocolitis (NEC), an acute hemorrhagic necrosis of thebowel which occurs in low birth weight infants and causes a significantmorbidity and mortality. Previous experiments have demonstrated thattreatment with glucocorticoids decreases the incidence of NEC in animalsand in premature infants, and the activity of glucocorticoids has beensuggested to occur via an increase in the activity of plasma PAF-AH.

A. Prevention of NEC

Recombinant PAF-AH (25,500 units in 0.3 ml, groups 2 and 4) orvehicle/buffer alone (25 mM tris, 0.5M NaCl, 1 mM EDTA and 0.1% Tween80) (groups 1 and 3) was administered into the tail veins of femaleWistar rats (n=3) weighing 180-220 grams. Either BSA (0.25%)-saline(groups 1 and 2) or PAF (0.2 μg/100 gm) suspended in BSA saline (groups3 and 4) was injected into the abdominal aorta at the level of thesuperior mesenteric artery 15 minutes after PAF-AH or vehicle injectionas previously described by Furukawa, et al. J. Pediatr.Res. 34:237-241(1993)!. The small intestines were removed after 2 hours from theligament of Trietz to the cecum, thoroughly washed with cold saline andexamined grossly. Samples were obtained from microscopic examinationfrom the upper, middle and lower portions of the small intestine. Thetissues were fixed in buffered formalin and the sample processed formicroscopic examination by staining with hematoxylin and eosin. Theexperiment was repeated three times.

Gross findings indicated a normal appearing bowel in groups treated withthe vehicle of BSA saline. Similarly, PAF-AH injected in the absence ofPAF had no effect on the gross findings. In contrast, the injection ofPAF into the descending aorta resulted in rapid, severe discolorationand hemorrhage of the serosal surface of the bowel. A similar hemorrhagewas noted when a section of the small bowel was examined on the mucosalside and the intestine appeared to be quite necrotic. When PAF-AH wasinjected via the tail vein 15 minutes prior to the administration of PAFinto the aorta the bowel appeared to be normal.

Upon microscopic examination, the intestine obtained from groups 1, 2and 4 demonstrated a normal villous architecture and a normal populationof cells within the lamina propria. In contrast, the group treated withPAF alone showed a full thickness necrosis and hemorrhage throughout theentire mucosa.

The plasma PAF-AH activities were also determined in the rats utilizedin the experiment described above. PAF-AH activity was determined asfollows. Prior to the tail vein injection, blood samples were obtained.Subsequently blood samples were obtained from the vena cava just priorto the injection of PAF and at the time of sacrifice. Approximately 50μl of blood was collected in heparinized capillaries. The plasma wasobtained following centrifugation (980×g for 5 minutes). The enzyme wasassayed as previously described by Yasuda and Johnston, Endocrinology,130:708-716 (1992).

The mean plasma PAF-AH activity of all rats prior to injection was foundto be 75.5±2.5 units (1 unit equals 1 nmoles×min⁻¹ ×ml⁻¹ plasma). Themean plasma PAF-AH activities 15 minutes following the injection of thevehicle were 75.2±2.6 units for group 1 and 76.7±3.5 units for group 3.After 15 minutes, the plasma PAF-AH activity of the animals injectedwith 25,500 units recombinant PAF-AH was 2249±341 units for group 2 and2494±623 units for group 4. The activity of groups 2 and 4 remainedelevated (1855±257 units) until the time of sacrifice (21/4 hours afterPAF-AH injection) (Group 2=1771±308; Group 4=1939±478). These resultsindicate that plasma PAF-AH activity of the rats which were injectedwith the vehicle alone (groups 1 and 3) did not change during the courseof the experiment. All the animals receiving the PAF injection alonedeveloped NEC while all rats that were injected with PAF-AH followed byPAF injection were completely protected.

B. Dose-Dependency of Prevention of NEC

In order to determine if the protection against NEC in rats was dosedependent, animals were treated with increasing doses of PAF-AH 15minutes prior to PAF administration. Initially, PAF-AH, ranging from25.5 to 25,500 units were administered into the tail vein of rats. PAF(0.4 μg in 0.2 ml of BSA-saline) was subsequently injected into theabdominal aorta 15 minutes after the administration of PAF-AH. The smallintestine was removed and examined for NEC development 2 hours after PAFadministration. Plasma PAF-AH activity was determined prior to theexogenous administration of the enzyme, and 15 minutes and 21/4 hoursafter PAF-AH administration. The results are the mean of 2-5 animals ineach group.

Gross findings indicated that all rats receiving less than 2,000 unitsof the enzyme developed NEC. Plasma PAF-AH activity in animals receivingthe lowest protective amount of enzyme (2040 units) was 363 units per mlof plasma after 15 minutes, representing a five-fold increase over basallevels. When PAF-AH was administered at less than 1,020 total units,resultant plasma enzyme activity averaged approximately 160 or less, andall animals developed NEC.

C. Duration of Protection Against NBC

In order to determine the length of time exogenous PAF-AH affordedprotection against development of NEC, rats were injected once with afixed amount of the enzyme via the tail vein and subsequently challengedwith PAF at various time points. PAF-AH (8,500 units in 0.3 ml) orvehicle alone was administered into the tail vein of rats, and PAF (0.36μg in 0.2 ml of BSA-saline) was injected into the abdominal aorta at thevarious times after the enzyme administration. The small intestines wereremoved 2 hours after the PAF injection for gross and histologicalexaminations in order to evaluate for NEC development. Plasma PAF-AHactivities were determined at various times after enzyme administrationand two hours after PAF administration. The mean value±standard errorfor enzyme activity was determined for each group.

Results indicated that none of the rats developed NEC within the firsteight hours after injection of PAF-AH, however 100% of the animalschallenged with PAF at 24 and 48 hours following injection of the enzymedeveloped NEC.

D. Reversal of NEC

In order to determine if administration of PAF-AH was capable ofreversing development of NEC induced by PAF injection, 25,500 units ofenzyme was administered via injection into the vena cava two minutesfollowing PAF administration (0.4 μg). None of the animals developedNEC. However, when PAF-AH was administered via this route 15 minutesafter the PAF injection, all animals developed NEC, consistent with therapid time course of NEC development as induced by the administration ofPAF previously reported Furukawa et al. supra!.

The sum of these observations indicate that a relatively small(five-fold) increase in the plasma PAF-AH activity is capable ofpreventing NEC. These observations combined with previous reports thatplasma PAF-AH activity in fetal rabbits Maki, et al.,Proc.Natl.Acad.Sci. (USA) 85:728-732 (1988)! and premature infantsCaplan, et al., J.Pediatr. 116:908-964 (1990)! has been demonstrated tobe relatively low suggests that prophylactic administration of humanrecombinant PAF-AH to low birth weight infants may be useful intreatment of NEC.

EXAMPLE 18

The efficacy of PAF-AH in a guinea pig model of acute respiratorydistress syndrome (ARDS) was examined.

Platelet-activating factor (PAF) injected intravenously into guinea pigsproduces a profound lung inflammation reminiscent of early ARDS inhumans. Within minutes after intravenous administration of PAF, the lungparenchyma becomes congested with constricted bronchi and bronchiolesLellouch-Tubiana et al., supra. Platelets and polymorphonuclearneutrophils begin to marginate and cellular aggregates are easilyidentified along arterioles of the lung Lellouch-Tubiana, Br. J. ExpPath., 66:345-355 (1985)!. PAF infusion also damages bronchialepithelial cells which dissociate from the airway walls and accumulatein the airway lumens. This damage to airway epithelial cells isconsistent with hyaline membrane formation that occurs in humans duringthe development of ARDS. Margination of the neutrophils and platelets isquickly followed by diapedesis of these cells into the alveolar septaand alveolar spaces of the lung. Cellular infiltrates elicited by PAFare accompanied by significant vascular leakage resulting in airwayedema Kirsch, Exp. Lung Res., 18:447-459 (1992)!. Evidence of edema isfurther supported by in vitro studies where PAF induces a dose-dependent(10-1000 ng/ml) extravasation of 125I labeled fibrinogen in perfusedguinea pig lungs Basran, Br. J. Pharmacol., 77:437 (1982)!.

Based on the above observations, an ARDS model in guinea pigs wasdeveloped. A cannula is placed into the jugular vein of anaesthetizedmale Hartly guinea pigs (approximately 350-400 grams) and PAF diluted ina 500 μl volume of phosphate buffered saline with 0.25% bovine serumalbumin as a carrier (PBS-BSA) is infused over a 15 minute period oftime at a total dosage ranging from 100-400 ng/kg. At various intervalsfollowing PAF infusion, animals are sacrificed and lung tissue iscollected. In guinea pigs infused with PAF, dose dependent lung damageand inflammation is clearly evident by 15 minutes and continues to bepresent at 60 minutes. Neutrophils and red blood cells are present inthe alveolar spaces of PAF treated guinea pigs but absent in control orsham infused animals. Evidence of epithelial cell damage is also evidentand reminiscent of hyaline membrane formation in human ARDS patients.Protein determinations done on bronchoalveolar lavage (BAL) samplestaken from guinea pigs infused with PAF shows a dramatic accumulation ofprotein in the inflamed lung, clear evidence of vascular leakage.

PAF-AH was found to completely protect against PAF mediated lung injuryin the guinea pig model of ARDS. Groups of guinea pigs were pretreatedwith either PAF-AH (2000 units in 500 μl) or 500 μl of the PAF-AH bufferonly. Fifteen minutes later these guinea pigs were infused with 400ng/kg PAF in a 500 μl volume, infused over a 15 minute period. Inaddition, a sham group of guinea pigs was infused with 500 μl ofPBS-BSA. At the completion of the PAF infusion the animals weresacrificed and BAL fluid was collected by lavaging the lungs 2× with10ml of saline containing 2μ/ml heparin to prevent clotting. Todetermine protein concentration in the BAL, samples were diluted 1:10 insaline and the OD 280 was determined. BAL fluid from sham guinea pigswas found to have a protein concentration of 2.10±1.3 mg/ml. In sharpcontrast, BAL fluid from animals infused with PAF was found to have aprotein concentration of 12.55±1.65 mg/ml. In guinea pigs pretreatedwith PAF-AH, BAL fluid was found to have a protein concentration of1.13±0.25 mg/ml which is comparable to the sham controls anddemonstrates that PAF-AH completely blocks lung edema in response toPAF.

EXAMPLE 19

Nearly four percent of the Japanese population has low or undetectablelevels of PAF-AH activity in their plasma. This deficiency has beencorrelated with severe respiratory symptoms in asthmatic children Miwaet al., J. Clin. Invest,. 82: 1983-1991 (1988)! who appear to haveinherited the deficiency in an autosomal recessive manner.

To determine if the deficiency arises from an inactive but presentenzyme or from an inability to synthesize PAF-AH, plasma from multiplepatients deficient in PAF-AH activity was assayed both for PAF-AHactivity (by the method described in Example 10 for transfectants) andfor the presence of PAF-AH using the monoclonal antibodies 90G11D and90F2D (Example 13) in a sandwich ELISA as follows. Immulon 4 flat bottomplates (Dynatech, Chantilly, Va.) were coated with 100 ng/well ofmonoclonal antibody 90G11D and stored overnight. The plates were blockedfor 1 hour at room temperature with 0.5% fish skin gelatin (Sigma)diluted in CMF-PBS and then washed three times. Patient plasma wasdiluted in PBS containing 15 mM CHAPS and added to each well of theplates (50 μl/well). The plates were incubated for 1 hour at roomtemperature and washed four times. Fifty μl of 5 μg/ml monoclonalantibody 90F2D, which was biotinylated by standard methods and dilutedin PBST, was added to each well, and the plates were incubated for 1hour at room temperature and then washed three times. Fifty μl ofExtraAvidin (Sigma) diluted 1/1000 in CMF-PBST was subsequently added toeach well and plates were incubated for 1 hour at room temperaturebefore development.

A direct correlation between PAF-AH activity and enzyme levels wasobserved. An absence of activity in a patient's serum was reflected byan absence of detectable enzyme. Similarly, plasma samples with half thenormal activity contained half the normal levels of PAF-AH. Theseobservations suggested that the deficiency of PAF-AH activity was due toan inability to synthesize the enzyme or due to an inactive enzyme whichthe monoclonal antibodies did not recognize.

Further experiments revealed that the deficiency was due to a geneticlesion in the human plasma PAF-AH gene. Genomic DNA from PAF-AHdeficient individuals was isolated and used as template for PCRreactions with PAF-AH gene specific primers. Each of the coding sequenceexons were initially amplified and sequenced from one individual. Asingle nucleotide change within exon 9 was observed (a G to T atposition 996 of SEQ ID NO:7). The nucleotide change results in an aminoacid substitution of a phenylalanine for a valine at position 279 of thePAF-AH sequence (V279F). Exon 9 was amplified from genomic DNA from anadditional eleven PAF-AH deficient individuals who were found to havethe same point mutation.

To test whether this mutation crippled the enzyme, an E. coli expressionconstruct containing the mutation was generated by methods similar tothat described in Example 10. When introduced into E. coli, theexpression construct generated no PAF-AH activity while a controlconstruct lacking the mutation was fully active. This amino acidsubstitution presumably results in a structural modification whichcauses the observed deficiency of activity and lack of immunoreactivitywith the PAF-AH antibodies of the invention.

PAF-AH specific antibodies of the invention may thus be used indiagnostic methods to detect abnormal levels of PAF-AH in serum (normallevels are about 1 to 5 U/ml) and to follow the progression of treatmentof pathological conditions with PAF-AH. Moreover, identification of agenetic lesion in the PAF-AH gene allows for genetic screening for thePAF-AH deficiency exhibited by the Japanese patients. The mutationcauses the gain of a restriction endonuclease site (Mae II) and thusallows for the simple method of Restriction Fragment Length Polymorphism(RFLP) analysis to differentiate between active and mutant alleles. SeeLewin, pp. 136-141 in Genes V, Oxford University Press, New York, N.Y.(1994).

Screening of genomic DNA from twelve PAF-AH deficient patients wascarried out by digestion of the DNA with MaeII, Southern blotting, andhybridization with an exon 9 probe (nucleotides 1-396 of SEQ ID NO:17).All patients were found to have RFLPs consistent with the mutant allele.

While the present invention has been described in terms of specificembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Accordingly, only such limitations asappear in the appended claims should be placed on the invention.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 36                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       PheLysAspLeuGlyGluGluAsnPheLysAlaLeuValLeuIleAla                              151015                                                                        Phe                                                                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       IleGlnValLeuMetAlaAlaAlaSerPheGlyGlnThrLysIlePro                              151015                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       MetLysProLeuValValPheValLeuGlyGly                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: group(13, 21, 27)                                               (C) OTHER INFORMATION: /note= "The nucleotide at each of                      these positions is an inosine."                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ACATGAATTCGGNATCYTTGNGTYTGNCCRAA32                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TATTTCTAGAAGTGTGGTGGAACTCGCTGG30                                              (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CGATGAATTCAGCTTGCAGCAGCCATCAGTAC32                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1520 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 162..1484                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GCTGGTCGGAGGCTCGCAGTGCTGTCGGCGAGAAGCAGTCGGGTTTGGAGCGCTTGGGTC60                GCGTTGGTGCGCGGTGGAACGCGCCCAGGGACCCCAGTTCCCGCGAGCAGCTCCGCGCCG120               CGCCTGAGAGACTAAGCTGAAACTGCTGCTCAGCTCCCAAGATGGTGCCACCC173                      MetValProPro                                                                  AAATTGCATGTGCTTTTCTGCCTCTGCGGCTGCCTGGCTGTGGTTTAT221                           LysLeuHisValLeuPheCysLeuCysGlyCysLeuAlaValValTyr                              5101520                                                                       CCTTTTGACTGGCAATACATAAATCCTGTTGCCCATATGAAATCATCA269                           ProPheAspTrpGlnTyrIleAsnProValAlaHisMetLysSerSer                              253035                                                                        GCATGGGTCAACAAAATACAAGTACTGATGGCTGCTGCAAGCTTTGGC317                           AlaTrpValAsnLysIleGlnValLeuMetAlaAlaAlaSerPheGly                              404550                                                                        CAAACTAAAATCCCCCGGGGAAATGGGCCTTATTCCGTTGGTTGTACA365                           GlnThrLysIleProArgGlyAsnGlyProTyrSerValGlyCysThr                              556065                                                                        GACTTAATGTTTGATCACACTAATAAGGGCACCTTCTTGCGTTTATAT413                           AspLeuMetPheAspHisThrAsnLysGlyThrPheLeuArgLeuTyr                              707580                                                                        TATCCATCCCAAGATAATGATCGCCTTGACACCCTTTGGATCCCAAAT461                           TyrProSerGlnAspAsnAspArgLeuAspThrLeuTrpIleProAsn                              859095100                                                                     AAAGAATATTTTTGGGGTCTTAGCAAATTTCTTGGAACACACTGGCTT509                           LysGluTyrPheTrpGlyLeuSerLysPheLeuGlyThrHisTrpLeu                              105110115                                                                     ATGGGCAACATTTTGAGGTTACTCTTTGGTTCAATGACAACTCCTGCA557                           MetGlyAsnIleLeuArgLeuLeuPheGlySerMetThrThrProAla                              120125130                                                                     AACTGGAATTCCCCTCTGAGGCCTGGTGAAAAATATCCACTTGTTGTT605                           AsnTrpAsnSerProLeuArgProGlyGluLysTyrProLeuValVal                              135140145                                                                     TTTTCTCATGGTCTTGGGGCATTCAGGACACTTTATTCTGCTATTGGC653                           PheSerHisGlyLeuGlyAlaPheArgThrLeuTyrSerAlaIleGly                              150155160                                                                     ATTGACCTGGCATCTCATGGGTTTATAGTTGCTGCTGTAGAACACAGA701                           IleAspLeuAlaSerHisGlyPheIleValAlaAlaValGluHisArg                              165170175180                                                                  GATAGATCTGCATCTGCAACTTACTATTTCAAGGACCAATCTGCTGCA749                           AspArgSerAlaSerAlaThrTyrTyrPheLysAspGlnSerAlaAla                              185190195                                                                     GAAATAGGGGACAAGTCTTGGCTCTACCTTAGAACCCTGAAACAAGAG797                           GluIleGlyAspLysSerTrpLeuTyrLeuArgThrLeuLysGlnGlu                              200205210                                                                     GAGGAGACACATATACGAAATGAGCAGGTACGGCAAAGAGCAAAAGAA845                           GluGluThrHisIleArgAsnGluGlnValArgGlnArgAlaLysGlu                              215220225                                                                     TGTTCCCAAGCTCTCAGTCTGATTCTTGACATTGATCATGGAAAGCCA893                           CysSerGlnAlaLeuSerLeuIleLeuAspIleAspHisGlyLysPro                              230235240                                                                     GTGAAGAATGCATTAGATTTAAAGTTTGATATGGAACAACTGAAGGAC941                           ValLysAsnAlaLeuAspLeuLysPheAspMetGluGlnLeuLysAsp                              245250255260                                                                  TCTATTGATAGGGAAAAAATAGCAGTAATTGGACATTCTTTTGGTGGA989                           SerIleAspArgGluLysIleAlaValIleGlyHisSerPheGlyGly                              265270275                                                                     GCAACGGTTATTCAGACTCTTAGTGAAGATCAGAGATTCAGATGTGGT1037                          AlaThrValIleGlnThrLeuSerGluAspGlnArgPheArgCysGly                              280285290                                                                     ATTGCCCTGGATGCATGGATGTTTCCACTGGGTGATGAAGTATATTCC1085                          IleAlaLeuAspAlaTrpMetPheProLeuGlyAspGluValTyrSer                              295300305                                                                     AGAATTCCTCAGCCCCTCTTTTTTATCAACTCTGAATATTTCCAATAT1133                          ArgIleProGlnProLeuPhePheIleAsnSerGluTyrPheGlnTyr                              310315320                                                                     CCTGCTAATATCATAAAAATGAAAAAATGCTACTCACCTGATAAAGAA1181                          ProAlaAsnIleIleLysMetLysLysCysTyrSerProAspLysGlu                              325330335340                                                                  AGAAAGATGATTACAATCAGGGGTTCAGTCCACCAGAATTTTGCTGAC1229                          ArgLysMetIleThrIleArgGlySerValHisGlnAsnPheAlaAsp                              345350355                                                                     TTCACTTTTGCAACTGGCAAAATAATTGGACACATGCTCAAATTAAAG1277                          PheThrPheAlaThrGlyLysIleIleGlyHisMetLeuLysLeuLys                              360365370                                                                     GGAGACATAGATTCAAATGTAGCTATTGATCTTAGCAACAAAGCTTCA1325                          GlyAspIleAspSerAsnValAlaIleAspLeuSerAsnLysAlaSer                              375380385                                                                     TTAGCATTCTTACAAAAGCATTTAGGACTTCATAAAGATTTTGATCAG1373                          LeuAlaPheLeuGlnLysHisLeuGlyLeuHisLysAspPheAspGln                              390395400                                                                     TGGGACTGCTTGATTGAAGGAGATGATGAGAATCTTATTCCAGGGACC1421                          TrpAspCysLeuIleGluGlyAspAspGluAsnLeuIleProGlyThr                              405410415420                                                                  AACATTAACACAACCAATCAACACATCATGTTACAGAACTCTTCAGGA1469                          AsnIleAsnThrThrAsnGlnHisIleMetLeuGlnAsnSerSerGly                              425430435                                                                     ATAGAGAAATACAATTAGGATTAAAATAGGTTTTTTAAAAAAAAAAAAAAA1520                       IleGluLysTyrAsn                                                               440                                                                           (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 441 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       MetValProProLysLeuHisValLeuPheCysLeuCysGlyCysLeu                              151015                                                                        AlaValValTyrProPheAspTrpGlnTyrIleAsnProValAlaHis                              202530                                                                        MetLysSerSerAlaTrpValAsnLysIleGlnValLeuMetAlaAla                              354045                                                                        AlaSerPheGlyGlnThrLysIleProArgGlyAsnGlyProTyrSer                              505560                                                                        ValGlyCysThrAspLeuMetPheAspHisThrAsnLysGlyThrPhe                              65707580                                                                      LeuArgLeuTyrTyrProSerGlnAspAsnAspArgLeuAspThrLeu                              859095                                                                        TrpIleProAsnLysGluTyrPheTrpGlyLeuSerLysPheLeuGly                              100105110                                                                     ThrHisTrpLeuMetGlyAsnIleLeuArgLeuLeuPheGlySerMet                              115120125                                                                     ThrThrProAlaAsnTrpAsnSerProLeuArgProGlyGluLysTyr                              130135140                                                                     ProLeuValValPheSerHisGlyLeuGlyAlaPheArgThrLeuTyr                              145150155160                                                                  SerAlaIleGlyIleAspLeuAlaSerHisGlyPheIleValAlaAla                              165170175                                                                     ValGluHisArgAspArgSerAlaSerAlaThrTyrTyrPheLysAsp                              180185190                                                                     GlnSerAlaAlaGluIleGlyAspLysSerTrpLeuTyrLeuArgThr                              195200205                                                                     LeuLysGlnGluGluGluThrHisIleArgAsnGluGlnValArgGln                              210215220                                                                     ArgAlaLysGluCysSerGlnAlaLeuSerLeuIleLeuAspIleAsp                              225230235240                                                                  HisGlyLysProValLysAsnAlaLeuAspLeuLysPheAspMetGlu                              245250255                                                                     GlnLeuLysAspSerIleAspArgGluLysIleAlaValIleGlyHis                              260265270                                                                     SerPheGlyGlyAlaThrValIleGlnThrLeuSerGluAspGlnArg                              275280285                                                                     PheArgCysGlyIleAlaLeuAspAlaTrpMetPheProLeuGlyAsp                              290295300                                                                     GluValTyrSerArgIleProGlnProLeuPhePheIleAsnSerGlu                              305310315320                                                                  TyrPheGlnTyrProAlaAsnIleIleLysMetLysLysCysTyrSer                              325330335                                                                     ProAspLysGluArgLysMetIleThrIleArgGlySerValHisGln                              340345350                                                                     AsnPheAlaAspPheThrPheAlaThrGlyLysIleIleGlyHisMet                              355360365                                                                     LeuLysLeuLysGlyAspIleAspSerAsnValAlaIleAspLeuSer                              370375380                                                                     AsnLysAlaSerLeuAlaPheLeuGlnLysHisLeuGlyLeuHisLys                              385390395400                                                                  AspPheAspGlnTrpAspCysLeuIleGluGlyAspAspGluAsnLeu                              405410415                                                                     IleProGlyThrAsnIleAsnThrThrAsnGlnHisIleMetLeuGln                              420425430                                                                     AsnSerSerGlyIleGluLysTyrAsn                                                   435440                                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1123 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: Not Determined                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       AAATATAAATTTTAATAACACCACACATAAATTTCAAACTACTTTCCCTAAGTTTCTAGC60                TGAAGTTTTAAATGAGTGTGTTTTTAATTTATTAGAAAGTGGATTGAAGAGAAAACATTG120               GAAGATGAAGGAAGGCGTTTCAGTTAAACCCCAAATAACTCTGTGTTACACTGAGCTATG180               AAACGGCTCCTTCTAGCTCCATTTCTCCTCAGACCTAAGTGCTATTCCTGATTGTCCTTC240               ATTGTCATTTCCAGGGAGAAATGACACCAGCACAGTGGCAGGCCTTCCAATCTGGAGCAC300               GGTCCACACAACTTCCGAATTGGTGTTCAGTGTAAAGTGTATCGGAGTGCGGAAAATGCG360               CAGGGCATTGCCAACTATAGATGCTCGGAGTAATTCAGTGTATTCAGAGAACACGGTGAA420               ACAAGGAAAACCGGCCTGACTGGGGGGTGAATTCAGCAGGGAGTAAATCTGATCGGCATC480               AGGTCTGCGGAAAGGAGCTGGTGAGCACGACACCACCAGGCATTGCCTGGCTCTCTCCGC540               GGCGGGCTAAGTTAACCTCGGGTCCAGGTGCGGGCCATGGTCTTGGGGAGGGTGCTGGGT600               GCGCTCGAGCAGGCTACGTCGGGAGCCGCCGCTGCTAGTGAGAGCCGGGCCACACACGCT660               CCTCCCCGGTACCTCCTCCAGCATCACCAGGGGAGGAGAGGGTCGGGCACAAGGCGCGCT720               AGGCGGACCCAGACACAGCCGCGCGCAGCCCACCCGCCCGCCGCCTGCCAGAGCTGCTCG780               GCCCGCAGCCAGGGGGACAGCGGCTGGTCGGAGGCTCGCAGTGCTGTCGGCGAGAAGCAG840               TCGGGTTTGGAGCGCTTGGGTCGCGTTGGTGCGCGGTGGAACCCCCCAGGGACCCCAGTT900               CCCGCGAGCAGCTCCGCGCCGCGCCTGAGTGAGGAGGGGCCCCGGGGGCGAGGCGGGAGT960               GGGAGGAAGGGCACGGTCGCCGCGCTGGAGGTCGGGACCCCGGAGCGGCGACCGGCCGGG1020              GTGGGCTCGCTGAGTCGCACCCGCTCTGCTGGCCGGTCCTGGGCTCACAGTCCCTGCAGC1080              CCTCGGAAACAGCGCTAGGATCCTTCGGGAGAGGAGAGATGAC1123                               (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 417 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 145..287                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GTACCAATCTAAAACCCAGCACAGAAAAATACATGTTTTATTTTTTCCAAGTGTTACTAG60                TACCTCAGCCTTTCTTGATTTGTCAGCTTATTTAAGGCCTCTTCATTGCATACTTCTTTT120               TTCTTTTAATCATCTGCTTCGAAGGAGACTAAGCTGAAACTGCTGCTCAGCTCCCAAGAT180               GGTGCCACCCAAATTGCATGTGCTTTTCTGCCTCTGCGGCTGCCTGGCTGTGGTTTATCC240               TTTTGACTGGCAATACATAAATCCTGTTGCCCATATGAAATCATCAGGTAAGAGGTGTAT300               TTGTTCAAGGTCTTGAGCAACTGATCTGTCGCCATACTTCAAGTGGGCCCCAAGAAGTTG360               CACATCTGCACATCTAAACAAGTCCTATTTAAAGGCTTATGGAGATCCTGTATTCTC417                  (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 498 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 251..372                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CATTAGGAGGTAACAGTCCAAGGCAGCTGAGAGAAAGGCTATGTCTACTTTCATCTCTTT60                ACCCTCCAAAACCCCTACACAGTGTTTCAAACAGAGAGACCCTCAATAATTGCATATCTT120               ACTTGTTAGGTTGAGAAAGAAAGAAGGCCAGAAACTATGGGAAGTAACTTGATTCCGTTG180               GAATTCTTTTGCATAATAAAATCTGATATGTAATGGATGACAAATGAGATAATATTTACC240               TGTTTTTCAGCATGGGTCAACAAAATACAAGTACTGATGGCTGCTGCAACGTTTGGCCAA300               ACTAAAATCCCCCGGGGAAATGGGCCTTATTCCGTTGGTTGTACAGACTTAATGTTTGAT360               CACACTAATAAGGTAATGCTTTGATTTATACAACTTATCCTGATACTCTAATATTGTCTG420               TCGCTATGGACCACTAGAAGGTGTTCAAATGTGACCTTGCCCTCACCTGAGAATGACTCA480               TTTTCGAATTTGTATTGT498                                                         (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 433 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 130..274                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      CAGCAGCCTAAAGTCTTAGACTTTGTGAACACAGAGGTATTGAGTCCCACTAATTAATAT60                CGAAAATAGCTGCTGGAATATGTTTGAGACACAACTTCTCTAAAAGTGCATTAATTTCTT120               TCTTAACAGGGCACCTTCTTGCGTTTATATTATCCATCCCAAGATAATGATCACCTTGAC180               ACCCTTTGGATCCCAAATAAAGAATATTTTTGGGGTCTTAGCAAATTTCTTGGAACACAC240               TGGCTTATGGGCAACATTTTGAGGTTACTCTTTGGTAAGATTTCTGTTGATCCTTCTTTG300               TAGGCTCTTGCATGTATGAAAACCTTGAAAACAACAAGAACTTCAAGTAGTTAAGACCAA360               AGTAGATTTTTCTTCAGTCCAAATAGCTCCTAAAATGATAAGGAAAGTATTTCTTTAAAG420               CCCAGGCAACTAC433                                                              (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 486 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 164..257                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      TTGGTGGGTATCTAGTAGCAGTCTTTTTAATGAATCTACTATTCATCCATAAAAAAGTAG60                ATATAAATCAGATGGGTCTGCATTTTATGCTAATGAGATATGAATTAAATTCACTAGCAA120               CACTCAGAGAAAACCTTAACTATAACCTTCCATTGTTGTCTAGGTTCAATGACAACTCCT180               GCAAACTGGAATTCCCCTCTGAGGCCTGGTGAAAAATATCCACTTGTTGTTTTTTCTCAT240               GGTCTTGGGGCATTCAGGTAATGTTTGAGAGGTTGAACAATTTTGGCTTCCAGGAATAAA300               TGACAATTTTTTTATTCAAGAAAGAAATAGCAGAGTTTGGAATGTCATGCAGGCCCTTGT360               CTGGAGGAGTTGGGGTTCCTCAATAATTGGCTGTGGGTCTATTGATCAGTCCTAGACCTG420               TCTGGTCAAGTAGTTTTTTCCCTACTATCAGCTCATTGGGATTAGCCTCACAGCAGAGAA480               GAAAGG486                                                                     (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 363 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 113..181                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      CCCCAGGCTCTACTACAGGGTGTAATGGCCTCCATGTTCCCAGTTTTATTAGTGACTCAG60                CCTTGTAATTCATGACTGGTAGTTGTAATTCTTCCCTCTTTTTGTTTTGAAGGACACTTT120               ATTCTGCTATTGGCATTGACCTGGCATCTCATGGGTTTATAGTTGCTGCTGTAGAACACA180               GGTATGTTACCTGATATAATTGGGCTCTTTGGCCAACTACAGGGAATGTCAATGCTCATA240               ACTATGTTTCTAATTTTCATAAAAGTTTATTTAAAATGTTGATGGAACTTTCAAGTATGG300               TAACATCATGAGCAAAAAAGGAGATTGAGTTTTATCGACTTAAAAGACTTAAAAGCACCT360               AAC363                                                                        (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 441 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 68..191                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GAACTGAGAAACATGGTCAGATGAGGAAGGGAAGGAGCATGCATAAATAATTTTGCTTGT60                ATTATAGAGATAGATCTGCATCTGCAACTTACTATTTCAAGGACCAATCTGCTGCAGAAA120               TAGGGGACAAGTCTTGGCTCTACCTTAGAACCCTGAAACAAGAGGAGGAGACACATATAC180               GAAATGAGCAGGTACATTGCAGTGAAAGGAGAGGTGGTTGGTGACCTAAAAGCATGTACA240               AAAGGATGACATTTGTTAATTTAATTTTACACCTGGCAAGTTATGCTCCTAGCTCTCCTA300               TTTCCCATTCCCAAAAGATCTGTCAATAGATTCCTGGAGCAGTAAAATTCCCTTAATGGA360               ATATCTAGTTCATAGTAAAAACAAAGGCAAATACAAAAATTTGGGAGATGACAGTGAATA420               TTCAGAATTCCTCGAGCCGGG441                                                      (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 577 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 245..358                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GGTTAAGTAAATCGTCTGAAGTCACATAGTAGGTAAGGCAAAACAGAGCCAGGATTTGGA60                CTAAGGCTATACCTATGTGCAAAGCTGGGGCCTGTGTCATTATGGTAGCAAGTAATAGTC120               ACTAATCAGATTTCCAGTTTATAACTGACCAACGATTTTTCCCAAATACAGCTTCTACCT180               AAACTTTAAAATAAGTGTTATAACTTTTTACTTTGTCATTTCCTTCTTCTAATAATTATA240               TTAGGTACGGCAAAGAGCAAAAGAATGTTCCCAAGCTCTCAGTCTGATTCTTGACATTGA300               TCATGGAAAGCCAGTGAAGAATGCATTAGATTTAAAGTTTGATATGGAACAACTGAAGGT360               AAGCTATAAAAAGTAATTTTTCTCTTGTCCTACAGTTCTTTATTGTTTTTTGTCATTTAA420               TTTTCTGCCTATATTGCAAGGTACAATATGATAAAGGGCTGCAACCAGCCCCTCCCCAAT480               GCGCACACACAGACACACAAAGCAGTACAGGTAAAGTATTGCAGCAATGAAGAATGCATT540               ATCTTGGACTAGATATGAATGCAAAGTTAGTCAGTTT577                                      (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 396 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 108..199                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      ATCAATGTATTTACCATCCCCATGAAATGAACAATTATATGATTGACAAATCATTTCTTC60                TAACACCACGAAATAGCTATAAATTTATATCATGCTTTTTCAAATAGGACTCTATTGATA120               GGGAAAAAATAGCAGTAATTGGACATTCTTTTGGTGGAGCAACGGTTATTCAGACTCTTA180               GTGAAGATCAGAGATTCAGGTAAGAAAATAAGATAGTAAAGCAAGAGAATAGTAAATTAT240               TGGAAGAAATTATATTGTGAGATATAATTTTTATTCAAATTCTTAGTGAAGGAAGGGGAT300               CTCTTGGAGTTTATAAGGCTATTCTTTTGCCCCCATAAAATACTCTATATACATTTTCCT360               AGGCTAAAACATCTCCTCTCCTGCTATTAAAATCTC396                                       (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 519 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 181..351                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      CTTACAAAGTTAATCATATCCCTTTCCCACATTGAAGTATGATACCTCTTTATTCCAATC60                AGATAACCCATAATAAACTGGTATGGTGCGTGTCCACCAATCCTAGCATTATTAGGATGT120               CCTCAATGTTGGCTAGTATGTAACCAGTTTAATTTCATCATTGTCAACAAATATCTACAG180               ATGTGGTATTGCCCTGGATGCATGGATGTTTCCACTGGGTGATGAAGTATATTCCAGAAT240               TCCTCAGCCCCTCTTTTTTATCAACTCTGAATATTTCCAATATCCTGCTAATATCATAAA300               AATGAAAAAATGCTACTCACCTGATAAAGAAAGAAAGATGATTACAATCAGGTAAGTATT360               AGTGACTTATTTCATTATGTGAAACAAACTTGAAGCTTGGGTAAATATCAATCGATATCA420               TTTGGTAACTATTAAAGAATTGCTGAATTGGTTGTTTAGACTTTCAATAAGGAGAGAATT480               AGATAATCTCAGTTTCTAAGTACATTTAGTCTACTCTTT519                                    (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 569 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 156..304                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      TGAAACACATCTAAGTAGATCAAATTACAAGTTTTATTTCTTCTTTGGTTTTCAGTAAAC60                AGACCAACAAGACCAGTACCTTTCCTTACACTCTAACTAAAAAAATAATAATTTTATCAA120               ACAATGTGACTTTTAAATGTCTTGTTCTCTTTTAGGGGTTCAGTCCACCAGAATTTTGCT180               GACTTCACTTTTGCAACTGGCAAAATAATTGGACACATGCTCAAATTAAAGGGAGACATA240               GATTCAAATGTAGCTATTGATCTTAGCAACAAAGCTTCATTAGCATTCTTACAAAAGCAT300               TTAGGTAAGAAACTATTTTTTTCATGACCTAAACCGAGATGAATCTCGAGGACAAAGCTG360               TCTATCTTAATACAGCTTTAGTACTATTTAAACTATTTCCAGTTGGTTTACAATGGAACA420               AAGCAGTATATCAATTTGAAAACAGAAATTTGAGAAAGTCAATTTTGCTGCTTTACATCT480               CTATATCATAGAAAGCAAATCAACTGTTAAAGGTAATATTCTTTGTATGAGCTAGAGTGA540               CTCATGTGAGGATATCGAACGACGGTGCT569                                              (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 469 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 137..253                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      GATACAGAGGCACATCGTCTCTACCATCCTAACGGAACTTGTGTAATTTGTAAATCTTTA60                TTGCCACCTAGGGGCATCCAAACTGTTTAATGCTCTCAAAAGTTTAATATGTTGATTAAC120               ACTTTATATTTTATAGGACTTCATAAAGATTTTGATCAGTGGGACTGCTTGATTGAAGGA180               GATGATGAGAATCTTATTCCAGGGACCAACATTAACACAACCAATCAACACATCATGTTA240               CAGAACTCTTCAGGAATAGAGAAATACAATTAGGATTAAAATAGGTTTTTTAAAAGTCTT300               GTTTCAAAACTGTCTAAAATTATGTGTGTGTGTGTGTGTGTGTGTGTGTGAGAGAGAGAG360               AGAGAGAGAGAGAGAGAATTTTAATGTATTTTCCCAAAGGACTCATATTTTAAAATGTAG420               GCTATACTGTAATCGTGATTGAAGCTTGGACTAAGAATTTTTTCCCTTT469                          (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1494 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 117..1436                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      GGCACGAGCTAGGATCTGACTCGCTCTGGTGGCATTGCTGCGCTCAGGGTTCTGGGTATC60                CGGGAGTCAGTGCAGTGACCAGAACATCAAACTGAAGCCACTGCTCAGCTCCTAAG116                   ATGGTACCACTCAAACTGCAGGCGCTTTTCTGCCTCCTCTGCTGCCTC164                           MetValProLeuLysLeuGlnAlaLeuPheCysLeuLeuCysCysLeu                              151015                                                                        CCATGGGTCCATCCTTTTCACTGGCAAGACACATCTTCTTTTGACTTC212                           ProTrpValHisProPheHisTrpGlnAspThrSerSerPheAspPhe                              202530                                                                        AGGCCGTCAGTAATGTTTCACAAGCTCCAATCGGTGATGTCTGCTGCC260                           ArgProSerValMetPheHisLysLeuGlnSerValMetSerAlaAla                              354045                                                                        GGCTCTGGCCATAGTAAAATCCCCAAAGGAAATGGATCGTACCCCGTC308                           GlySerGlyHisSerLysIleProLysGlyAsnGlySerTyrProVal                              505560                                                                        GGTTGTACAGATCTGATGTTCGGTTATGGGAATGAGAGCGTCTTCGTG356                           GlyCysThrAspLeuMetPheGlyTyrGlyAsnGluSerValPheVal                              65707580                                                                      CGTTTGTACTACCCAGCTCAAGATCAAGGTCGCCTCGACACTGTTTGG404                           ArgLeuTyrTyrProAlaGlnAspGlnGlyArgLeuAspThrValTrp                              859095                                                                        ATCCCAAACAAAGAATATTTTTTGGGTCTTAGTATATTTCTTGGAACA452                           IleProAsnLysGluTyrPheLeuGlyLeuSerIlePheLeuGlyThr                              100105110                                                                     CCCAGTATTGTAGGCAATATTTTACACCTCTTATATGGTTCTCTGACA500                           ProSerIleValGlyAsnIleLeuHisLeuLeuTyrGlySerLeuThr                              115120125                                                                     ACTCCTGCAAGCTGGAATTCTCCTTTAAGGACTGGAGAAAAATACCCG548                           ThrProAlaSerTrpAsnSerProLeuArgThrGlyGluLysTyrPro                              130135140                                                                     CTCATTGTCTTTTCTCATGGTCTCGGAGCCTTCAGGACGATTTATTCT596                           LeuIleValPheSerHisGlyLeuGlyAlaPheArgThrIleTyrSer                              145150155160                                                                  GCTATTGGCATTGGCTTGGCATCTAATGGGTTTATAGTGGCCACTGTC644                           AlaIleGlyIleGlyLeuAlaSerAsnGlyPheIleValAlaThrVal                              165170175                                                                     GAACACAGAGACAGATCTGCATCGGCAACTTACTTTTTTGAAGACCAG692                           GluHisArgAspArgSerAlaSerAlaThrTyrPhePheGluAspGln                              180185190                                                                     GTGGCTGCAAAAGTGGAAAACAGGTCTTGGCTTTACCTGAGAAAAGTA740                           ValAlaAlaLysValGluAsnArgSerTrpLeuTyrLeuArgLysVal                              195200205                                                                     AAACAAGAGGAGTCGGAAAGTGTCCGGAAAGAACAGGTTCAGCAAAGA788                           LysGlnGluGluSerGluSerValArgLysGluGlnValGlnGlnArg                              210215220                                                                     GCAATAGAATGTTCCCGGGCTCTCAGTGCGATTCTTGACATTGAACAT836                           AlaIleGluCysSerArgAlaLeuSerAlaIleLeuAspIleGluHis                              225230235240                                                                  GGAGACCCAAAAGAGAATGTACTAGGTTCAGCTTTTGACATGAAACAG884                           GlyAspProLysGluAsnValLeuGlySerAlaPheAspMetLysGln                              245250255                                                                     CTGAAGGATGCTATTGATGAGACTAAAATAGCTTTGATGGGACATTCT932                           LeuLysAspAlaIleAspGluThrLysIleAlaLeuMetGlyHisSer                              260265270                                                                     TTTGGAGGAGCAACAGTTCTTCAAGCCCTTAGTGAGGACCAGAGATTC980                           PheGlyGlyAlaThrValLeuGlnAlaLeuSerGluAspGlnArgPhe                              275280285                                                                     AGATGTGGAGTTGCTCTTGATCCATGGATGTATCCGGTGAACGAAGAG1028                          ArgCysGlyValAlaLeuAspProTrpMetTyrProValAsnGluGlu                              290295300                                                                     CTGTACTCCAGAACCCTCCAGCCTCTCCTCTTTATCAACTCTGCCAAA1076                          LeuTyrSerArgThrLeuGlnProLeuLeuPheIleAsnSerAlaLys                              305310315320                                                                  TTCCAGACTCCAAAGGACATCGCAAAAATGAAAAAGTTCTACCAGCCT1124                          PheGlnThrProLysAspIleAlaLysMetLysLysPheTyrGlnPro                              325330335                                                                     GACAAGGAAAGGAAAAATGATTACAATCAAGGGCTCAGGCACCAGAAC1172                          AspLysGluArgLysAsnAspTyrAsnGlnGlyLeuArgHisGlnAsn                              340345350                                                                     TTTGACGACTTTACTTTTGTAACTGGCAAAATAATTGGAAACAAGCTG1220                          PheAspAspPheThrPheValThrGlyLysIleIleGlyAsnLysLeu                              355360365                                                                     ACACTGAAAGGAGAAATCGATTCCAGAGTAGCCATCGACCTCACCAAC1268                          ThrLeuLysGlyGluIleAspSerArgValAlaIleAspLeuThrAsn                              370375380                                                                     AAAGCTTCGATGGCTTTCTTACAAAAGCATTTAGGGCTTCAGAAAGAC1316                          LysAlaSerMetAlaPheLeuGlnLysHisLeuGlyLeuGlnLysAsp                              385390395400                                                                  TTTGATCAGTGGGACCCTCTGGTGGAAGGAGATGATGAGAACCTGATT1364                          PheAspGlnTrpAspProLeuValGluGlyAspAspGluAsnLeuIle                              405410415                                                                     CCTGGGTCACCCTTTGACGCAGTCACCCAGGCCCCGGCTCAGCAACAC1412                          ProGlySerProPheAspAlaValThrGlnAlaProAlaGlnGlnHis                              420425430                                                                     TCTCCAGGATCACAGACCCAGAATTAGAAGAACTTGCTTGTTACACAGTTGCCT1466                    SerProGlySerGlnThrGlnAsn                                                      435440                                                                        TTTAAAAGTAGAGTGACATGAGAGAGAG1494                                              (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2191 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 92..1423                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      CCGCGCGCTCCGGCCGGGGGACCCTGGTTCCGGCGAGCGGCTCAGCGCGGCGCCCGGAAG60                TTTAAGCTGAAACCACTGCTCAGCTTCCAAGATGTTGCCACCCAAACTGCAT112                       MetLeuProProLysLeuHis                                                         15                                                                            GCGCTTTTCTGCCTCTGCAGCTGCCTCACACTGGTTCATCCTATTGAC160                           AlaLeuPheCysLeuCysSerCysLeuThrLeuValHisProIleAsp                              101520                                                                        TGGCAAGACCTAAATCCTGTTGCCCATATTAGATCATCAGCATGGGCC208                           TrpGlnAspLeuAsnProValAlaHisIleArgSerSerAlaTrpAla                              253035                                                                        AATAAAATACAAGCTCTGATGGCTGCTGCAAGTATTAGGCAAAGTAGA256                           AsnLysIleGlnAlaLeuMetAlaAlaAlaSerIleArgGlnSerArg                              40455055                                                                      ATTCCCAAAGGAAATGGATCTTATTCTGTCGGTTGTACAGATTTGATG304                           IleProLysGlyAsnGlySerTyrSerValGlyCysThrAspLeuMet                              606570                                                                        TTTGATTATACTAATAAGGGCACCTTTTTGCGTTTGTATTATCCATCG352                           PheAspTyrThrAsnLysGlyThrPheLeuArgLeuTyrTyrProSer                              758085                                                                        CAAGAGGATGACCACTCTGACACGCTTTGGATCCCAAACAAAGAATAT400                           GlnGluAspAspHisSerAspThrLeuTrpIleProAsnLysGluTyr                              9095100                                                                       TTTTTTGGTCTTAGTAAATATCTTGGAACACCCTGGCTTATGGGCAAA448                           PhePheGlyLeuSerLysTyrLeuGlyThrProTrpLeuMetGlyLys                              105110115                                                                     ATATTGAGCTTCTTTTTTGGTTCAGTGACAACTCCTGCGAACTGGAAT496                           IleLeuSerPhePhePheGlySerValThrThrProAlaAsnTrpAsn                              120125130135                                                                  TCCCCTCTGAGGACTGGTGAAAAATATCCACTGATTGTTTTTTCTCAT544                           SerProLeuArgThrGlyGluLysTyrProLeuIleValPheSerHis                              140145150                                                                     GGTCTTGGAGCATTCCGGACAATTTATTCTGCTATTGGCATTGATCTA592                           GlyLeuGlyAlaPheArgThrIleTyrSerAlaIleGlyIleAspLeu                              155160165                                                                     GCATCACATGGGTTCATCGTTGCTGCTATAGAACACAGAGATGGATCC640                           AlaSerHisGlyPheIleValAlaAlaIleGluHisArgAspGlySer                              170175180                                                                     GCCTCTGCGACTTACTATTTCAAGGACCAGTCTGCTGCAGAAATAGGG688                           AlaSerAlaThrTyrTyrPheLysAspGlnSerAlaAlaGluIleGly                              185190195                                                                     AACAAATCTTGGTCTTATCTTCAAGAACTAAAACCAGGGGATGAGGAG736                           AsnLysSerTrpSerTyrLeuGlnGluLeuLysProGlyAspGluGlu                              200205210215                                                                  ATACATGTTCGAAATGAGCAGGTACAGAAAAGGGCAAAGGAGTGCTCC784                           IleHisValArgAsnGluGlnValGlnLysArgAlaLysGluCysSer                              220225230                                                                     CAAGCTCTCAACTTGATTCTGGACATTGATCATGGAAGGCCAATTAAG832                           GlnAlaLeuAsnLeuIleLeuAspIleAspHisGlyArgProIleLys                              235240245                                                                     AATGTACTAGACTTAGAGTTTGATGTGGAACAACTGAAGGACTCTATT880                           AsnValLeuAspLeuGluPheAspValGluGlnLeuLysAspSerIle                              250255260                                                                     GACAGGGATAAAATAGCAGTAATTGGACATTCTTTTGGTGGAGCCACA928                           AspArgAspLysIleAlaValIleGlyHisSerPheGlyGlyAlaThr                              265270275                                                                     GTTCTTCAGGCTCTTAGTGAAGACCAGAGATTTAGGTGCGGGATTGCC976                           ValLeuGlnAlaLeuSerGluAspGlnArgPheArgCysGlyIleAla                              280285290295                                                                  TTGGATGCATGGATGCTTCCACTGGATGATGCAATATATTCCAGAATC1024                          LeuAspAlaTrpMetLeuProLeuAspAspAlaIleTyrSerArgIle                              300305310                                                                     CCTCAGCCCCTCTTTTTTATTAACTCGGAACGGTTCCAATTTCCTGAG1072                          ProGlnProLeuPhePheIleAsnSerGluArgPheGlnPheProGlu                              315320325                                                                     AATATCAAAAAAATGAAAAAATGCTACTCACCTGACAAAGAAAGAAAA1120                          AsnIleLysLysMetLysLysCysTyrSerProAspLysGluArgLys                              330335340                                                                     ATGATTACAATCAGGGGTTCAGTCCATCAGAACTTTGCTGATTTCACT1168                          MetIleThrIleArgGlySerValHisGlnAsnPheAlaAspPheThr                              345350355                                                                     TTTACAACTGGCAAAATAGTTGGATACATATTCACATTAAAAGGAGAT1216                          PheThrThrGlyLysIleValGlyTyrIlePheThrLeuLysGlyAsp                              360365370375                                                                  ATAGATTCAAATGTAGCAATTGATCTTTGCAACAAAGCTTCATTGGCA1264                          IleAspSerAsnValAlaIleAspLeuCysAsnLysAlaSerLeuAla                              380385390                                                                     TTTTTACAAAAGCATTTAGGACTGCGGAAAGATTTTGATCAGTGGGAT1312                          PheLeuGlnLysHisLeuGlyLeuArgLysAspPheAspGlnTrpAsp                              395400405                                                                     TCTTTGATTGAAGGAAAAGACGAAAATCTTATGCCAGGGACCAACATT1360                          SerLeuIleGluGlyLysAspGluAsnLeuMetProGlyThrAsnIle                              410415420                                                                     AACATCACCAACGAACATGACACTCTACAGAACTCTCCAGAAGCAGAG1408                          AsnIleThrAsnGluHisAspThrLeuGlnAsnSerProGluAlaGlu                              425430435                                                                     AAATCGAATTTAGATTAAAAGCACTTTTTTAAAGATCTTGTTTAAAAACTGTCAA1463                   LysSerAsnLeuAsp                                                               440                                                                           AAAATGTGTGTATGACTTTTAATATATTTTCTCAAATAACTCATATTGGAAAATGTAGGC1523              TATCCCATAAAAGTGATTGAAGCTTGGACTAGGAGGTTTTTTTCTTTAAAGAAAGATTGG1583              TGTCTATCGAAATCATGCCAGCCTAAATTTTAATTTTACTAAAATGATGCTGTGTCAAAA1643              TTAATAACTACTTTTACATTCTTTAATGGACAAGTATAACAGGCACAAGGCTAATGAAAA1703              CGTGTTGCAATGACATAACAATCCCTAAAAATACAGATGTTCTTGCCTCTTTTTTCTATT1763              ATAATTGAGTTTTAGCAACATGTTATGCTAGGTAGAATTTGGAAGCACTTCCCTTTGACT1823              TTTGGTCATGATAAGAAAAATTAGATCAAGCAAATGATAAAAGCAGTGTTTTACCAAGGA1883              TTAGGGATACTGAACAATTTCACTATGGTAACTGAATGGGGAGTGACCAAGGGTAAAAAT1943              ATTAAAGCCAAGGCAAAGGCAGCAGATTAGAATGGATTAAAGAGAGTTTATAATTTGTTT2003              GCATTTACTTGATGGTTTATCTCATGGATTCATGAGTCAAGAAAGGTGCGTAGGACAGGC2063              CAGGGATTCCAGTTATAACACATTATTCACCCAAAGGGTTCTTTAATTCTGTATGAGTAT2123              TGGGAGTGGATTAGCACAATAGAGGCATATGTTGCTTTAAAAAAAAAAAAAAAAAAAAAA2183              AAAAAAAA2191                                                                  (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1533 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 62..1394                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      CCGCGAGCAGTTCACCGCGGCGTCCGGAAGGTTAAGCTGAAACGGCAGCTCAGCTTCGGA60                GATGTTACCGTCCAAATTGCATGCGCTTTTCTGCCTCTGCACCTGC106                             MetLeuProSerLysLeuHisAlaLeuPheCysLeuCysThrCys                                 151015                                                                        CTTGCACTGGTTTATCCTTTTGACTGGCAAGACCTGAATCCAGTTGCC154                           LeuAlaLeuValTyrProPheAspTrpGlnAspLeuAsnProValAla                              202530                                                                        TATATTGAATCACCAGCATGGGTCAGTAAGATACAAGCTCTGATGGCT202                           TyrIleGluSerProAlaTrpValSerLysIleGlnAlaLeuMetAla                              354045                                                                        GCTGCAAACATTGGTCAATCTAAAATCCCCAGAGGAAATGGATCTTAT250                           AlaAlaAsnIleGlyGlnSerLysIleProArgGlyAsnGlySerTyr                              505560                                                                        TCCGTCGGTTGTACAGACTTGATGTTTGATTACACTAATAAGGGCACC298                           SerValGlyCysThrAspLeuMetPheAspTyrThrAsnLysGlyThr                              657075                                                                        TTCTTGCGTTTGTATTATCCATCTCAAGATGATGATCACTCCGACACC346                           PheLeuArgLeuTyrTyrProSerGlnAspAspAspHisSerAspThr                              80859095                                                                      CTTTGGATCCCAAACAAAGAATATTTTTTGGGTCTTAGTAAATTTCTT394                           LeuTrpIleProAsnLysGluTyrPheLeuGlyLeuSerLysPheLeu                              100105110                                                                     GGAACACACTGGCTTGTGGGCAAAATTATGGGCTTATTCTTCGGTTCA442                           GlyThrHisTrpLeuValGlyLysIleMetGlyLeuPhePheGlySer                              115120125                                                                     ATGACAACTCCTGCAGCCTGGAATGCACATCTGAGGACTGGGGAAAAA490                           MetThrThrProAlaAlaTrpAsnAlaHisLeuArgThrGlyGluLys                              130135140                                                                     TACCCACTAATTATTTTTTCTCATGGTCTTGGAGCATTCAGGACGATT538                           TyrProLeuIleIlePheSerHisGlyLeuGlyAlaPheArgThrIle                              145150155                                                                     TATTCTGCTATTGGCATTGATCTGGCATCCCACGGGTTTATAGTTGCT586                           TyrSerAlaIleGlyIleAspLeuAlaSerHisGlyPheIleValAla                              160165170175                                                                  GCTGTAGAACACAGGGATGGCTCTGCATCCTCGACATACTATTTCAAG634                           AlaValGluHisArgAspGlySerAlaSerSerThrTyrTyrPheLys                              180185190                                                                     GACCAGTCTGCTGTAGAAATAGGCAACAAGTCTTGGCTCTATCTCAGA682                           AspGlnSerAlaValGluIleGlyAsnLysSerTrpLeuTyrLeuArg                              195200205                                                                     ACCCTGAAGCGAGGAGAGGAGGAGTTTCCTTTACGAAATGAGCAGTTA730                           ThrLeuLysArgGlyGluGluGluPheProLeuArgAsnGluGlnLeu                              210215220                                                                     CGGCAACGAGCAAAGGAATGTTCTCAAGCTCTCAGTTTGATTCTGGAC778                           ArgGlnArgAlaLysGluCysSerGlnAlaLeuSerLeuIleLeuAsp                              225230235                                                                     ATTGATCACGGGAGGCCAGTGACGAATGTACTAGATTTAGAGTTTGAT826                           IleAspHisGlyArgProValThrAsnValLeuAspLeuGluPheAsp                              240245250255                                                                  GTGGAACAGCTGAAGGACTCTATTGATAGGGATAAAATAGCCATTATT874                           ValGluGlnLeuLysAspSerIleAspArgAspLysIleAlaIleIle                              260265270                                                                     GGACATTCTTTTGGTGGAGCCACAGTTATTCAGACTCTTAGTGAAGAC922                           GlyHisSerPheGlyGlyAlaThrValIleGlnThrLeuSerGluAsp                              275280285                                                                     CAGAGATTCAGGTGTGGCATTGCTCTGGATGCATGGATGTTTCCCGTG970                           GlnArgPheArgCysGlyIleAlaLeuAspAlaTrpMetPheProVal                              290295300                                                                     GGTGATGAAGTATATTCCAGAATTCCTCAACCCCTCTTTTTTATCAAC1018                          GlyAspGluValTyrSerArgIleProGlnProLeuPhePheIleAsn                              305310315                                                                     TCGGAACGATTCCAATACCCTTCTAATATCATAAGAATGAAAAAATGC1066                          SerGluArgPheGlnTyrProSerAsnIleIleArgMetLysLysCys                              320325330335                                                                  TTCTTACCTGATAGAGAACGAAAAATGATTACAATCAGGGGTTCGGTC1114                          PheLeuProAspArgGluArgLysMetIleThrIleArgGlySerVal                              340345350                                                                     CATCAGAATTTTGTTGACTTCACTTTTGCCACTAGCAAAATAATTGGC1162                          HisGlnAsnPheValAspPheThrPheAlaThrSerLysIleIleGly                              355360365                                                                     TACCTATTCACACTGAAAGGAGACATCGATTCCAATGTAGCCATCAGC1210                          TyrLeuPheThrLeuLysGlyAspIleAspSerAsnValAlaIleSer                              370375380                                                                     CTTAGCAACAAAGCTTCCTTAGCGTTCTTACAAAAACATTTAGGACTT1258                          LeuSerAsnLysAlaSerLeuAlaPheLeuGlnLysHisLeuGlyLeu                              385390395                                                                     CAGAAAGATTTTGATCAGTGGGATTCTTTAGTTGAAGGCGAAGATCAC1306                          GlnLysAspPheAspGlnTrpAspSerLeuValGluGlyGluAspHis                              400405410415                                                                  AATCTTATTCCAGGGACCAACATTAACACAACCAACCACCAAGCCATT1354                          AsnLeuIleProGlyThrAsnIleAsnThrThrAsnHisGlnAlaIle                              420425430                                                                     CTGCAGAACTCCACAGGAATAGAGAGACCAAATTTAGATTAAAAGAGCTT1404                        LeuGlnAsnSerThrGlyIleGluArgProAsnLeuAsp                                       435440                                                                        TTTAAAAAGTTTTGTTTACGAACTTGTCTAAAAGTGTGTGTGTGTATGATTTAAATGTAT1464              TTTCTCAAATAGCTCATATTAAAAAATGTAGGCTATAGCACAAAAAAAAAAAAAAAAAAA1524              AAAAAAAAA1533                                                                 (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1876 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 468..1734                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      CGGCGGGCTGCTGGCCCTTCCCGGCTGTTCGTAGAGCCGGATCCTGCAGCGCCCCTGAGA60                CGAACCGCCCCGATGCGGTGCTCCTCAGCGCCACGGGACGCAGCCGGGGCCGGCCGTGTT120               GGCGCAGCTCCCACGACGTACGCTTCCTTTCCAGGCTCGAGGAAAGCCTCTCCCACAAAC180               ACCGTCCCAGCTGGGAAGTGAGGCGGAGTTTTGGTCCCTCCCCTCCGGCAGCGCCCGGCA240               TTCCGTCCGTCCGTCCGTCCGTCCGTGCGGCGCACGGCGCCCTGCAGAGCCGGGACACCG300               CAGCAGGGTAGGAGGACCCGGAGGTGGTGTGCAGCCACAGGTTTCCATCCTGCCCCCACC360               TCCCGGGGAGCAGCCCTGTGCTATACCCAACCCCCCGCACAGAGCACTGAGCCGGCTGCT420               GCCTGCCTGCACCCCGCCGTGGGACCTTCTGCTCTTCCCAACAAGTGATGGCATCG476                   MetAlaSer                                                                     1                                                                             CTGTGGGTGAGAGCCAGGAGGGTGTTCATGAAAAGTCGTGCTTCAGGT524                           LeuTrpValArgAlaArgArgValPheMetLysSerArgAlaSerGly                              51015                                                                         TTCTCGGCGAAGGCGGCGACGGAGATGGGGAGCGGCGGCGCGGAGAAG572                           PheSerAlaLysAlaAlaThrGluMetGlySerGlyGlyAlaGluLys                              20253035                                                                      GGCTATCGGATCCCCGCCGGGAAGGGCCCGCACGCCGTGGGCTGCACG620                           GlyTyrArgIleProAlaGlyLysGlyProHisAlaValGlyCysThr                              404550                                                                        GATCTGATGACCGGCGACGCGGCCGAGGGAAGCTTTTTGCGCCTGTAT668                           AspLeuMetThrGlyAspAlaAlaGluGlySerPheLeuArgLeuTyr                              556065                                                                        TACCTATCGTGTGACGACACAGATACTGAAGAGACACCCTGGATTCCA716                           TyrLeuSerCysAspAspThrAspThrGluGluThrProTrpIlePro                              707580                                                                        GATAAAGAGTACTACCAGGGGCTGTCTGACTTCCTCAACGTGTACCGG764                           AspLysGluTyrTyrGlnGlyLeuSerAspPheLeuAsnValTyrArg                              859095                                                                        GCCCTGGGAGAAAGGCTTTTCCAGTACTACGTTGGCTCAGTGACCTGT812                           AlaLeuGlyGluArgLeuPheGlnTyrTyrValGlySerValThrCys                              100105110115                                                                  CCTGCAAAATCAAACGCTGCTTTTAAGCCAGGAGAGAAATACCCACTG860                           ProAlaLysSerAsnAlaAlaPheLysProGlyGluLysTyrProLeu                              120125130                                                                     CTCGTTTTTTCCCATGGACTTGGAGCTTTTCGGACCATCTATTCTGCT908                           LeuValPheSerHisGlyLeuGlyAlaPheArgThrIleTyrSerAla                              135140145                                                                     ATCTGCATAGAGATGGCTTCTCAAGGCTTTCTAGTGGCAGCTGTGGAG956                           IleCysIleGluMetAlaSerGlnGlyPheLeuValAlaAlaValGlu                              150155160                                                                     CACAGAGATGAATCGGCTTCAGCAACGTATTTCTGTAAAAAGAAGGCT1004                          HisArgAspGluSerAlaSerAlaThrTyrPheCysLysLysLysAla                              165170175                                                                     GATTCTGAGCCAGAGGAGGATCAAACATCAGGCGTGGAGAAGGAGTGG1052                          AspSerGluProGluGluAspGlnThrSerGlyValGluLysGluTrp                              180185190195                                                                  ATCTACTACAGGAAGCTCAGAGCAGGAGAGGAGGAGCGCTGTCTGCGT1100                          IleTyrTyrArgLysLeuArgAlaGlyGluGluGluArgCysLeuArg                              200205210                                                                     CACAAGCAGGTACAGCAGAGAGCACAGGAGTGCATCAAAGCGCTCAAC1148                          HisLysGlnValGlnGlnArgAlaGlnGluCysIleLysAlaLeuAsn                              215220225                                                                     CTCATTCTTAAGATCAGTTCAGGAGAGGAAGTGATGAATGTGCTGAAC1196                          LeuIleLeuLysIleSerSerGlyGluGluValMetAsnValLeuAsn                              230235240                                                                     TCAGACTTTGACTGGAACCACCTGAAGGATTCTGTTGATACTAGCAGA1244                          SerAspPheAspTrpAsnHisLeuLysAspSerValAspThrSerArg                              245250255                                                                     ATAGCTGTGATGGGACACTCTTTTGGTGGTGCTACAGTTATTGAGAGC1292                          IleAlaValMetGlyHisSerPheGlyGlyAlaThrValIleGluSer                              260265270275                                                                  CTCAGCAAAGAAATTAGATTTAGGTGTGGCATTGCCCTTGATGCGTGG1340                          LeuSerLysGluIleArgPheArgCysGlyIleAlaLeuAspAlaTrp                              280285290                                                                     ATGCTCCCGGTAGGCGATGACACTTACCAAAGCAGTGTGCAGCAACCA1388                          MetLeuProValGlyAspAspThrTyrGlnSerSerValGlnGlnPro                              295300305                                                                     CTGCTCTTTATTAATTCCGAAAAATTCCAGTGGGCTGCCAATATCTTA1436                          LeuLeuPheIleAsnSerGluLysPheGlnTrpAlaAlaAsnIleLeu                              310315320                                                                     AAGATGAAGAAGCTTAGCTCCAATGATACCAACAAGAAAATGATCACC1484                          LysMetLysLysLeuSerSerAsnAspThrAsnLysLysMetIleThr                              325330335                                                                     ATCAAAGGATCGGTACATCAGAGCTTTCCTGATTTTACTTTTGTGAGT1532                          IleLysGlySerValHisGlnSerPheProAspPheThrPheValSer                              340345350355                                                                  GGAGAAATCATTGGAAAGTTTTTCAAGTTAAAAGGAGAAATAGACCCA1580                          GlyGluIleIleGlyLysPhePheLysLeuLysGlyGluIleAspPro                              360365370                                                                     AATGAAGCTATTGATATATGCAACCACGCTTCATTGGCCTTCCTGCAG1628                          AsnGluAlaIleAspIleCysAsnHisAlaSerLeuAlaPheLeuGln                              375380385                                                                     AAACATCTGAGTCTTAAGAGAGATTTTGATAAGTGGGATTCACTCGTG1676                          LysHisLeuSerLeuLysArgAspPheAspLysTrpAspSerLeuVal                              390395400                                                                     GATGGCATAGGACCCAATGTTATTTCTGGTACCAATATCGACTTATCT1724                          AspGlyIleGlyProAsnValIleSerGlyThrAsnIleAspLeuSer                              405410415                                                                     CCAACTGAGTAAGGAGTACAAGAAGTACTGCAAAGGCCACCAGCAGCAGG1774                        ProThrGlu                                                                     420                                                                           ACACCAACGTTGGCCACACATTGCTTGGAGCTGAGATAGCACTGGCCTCCCACACAGCTT1834              TTGGAGTGTGAAACAACAAAAAAAAAAATCACAGGGGAGCCG1876                                (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 517 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 2..514                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      GGGGCATTCTTTTGGAGGAGCAACAGTTTTTCAAGCCCTAAGTGAA46                              GlyHisSerPheGlyGlyAlaThrValPheGlnAlaLeuSerGlu                                 151015                                                                        GACCAGAGATTCAGATGTGGGATTGCCCTTGATCCGTGGATGTTTCCC94                            AspGlnArgPheArgCysGlyIleAlaLeuAspProTrpMetPhePro                              202530                                                                        GTGAGTGAGGAGCTGTACTCCAGAGTTCCTCAGCCTCTCTTCTTTATC142                           ValSerGluGluLeuTyrSerArgValProGlnProLeuPhePheIle                              354045                                                                        AACTCTGCCGAATTCCAGACTCCAAAGGACATTGCAAAAATGAAAAAC190                           AsnSerAlaGluPheGlnThrProLysAspIleAlaLysMetLysAsn                              505560                                                                        TTCTACCAGCCTGACAAGGAAAGGAAAATGATTACGATCAAGGGCTCA238                           PheTyrGlnProAspLysGluArgLysMetIleThrIleLysGlySer                              657075                                                                        GTGCACCAGAATTTTGCTGACGGGACTTTTGTAACTGGCAAAATAATT286                           ValHisGlnAsnPheAlaAspGlyThrPheValThrGlyLysIleIle                              80859095                                                                      GGAAACAAGCTGTCACTGAAAGGAGACATAGACTCCAGAGTTGCCATA334                           GlyAsnLysLeuSerLeuLysGlyAspIleAspSerArgValAlaIle                              100105110                                                                     GACCTCACCAACAAGGCTTCCTTGGCTTTCTTACAAAAACATTTAGGA382                           AspLeuThrAsnLysAlaSerLeuAlaPheLeuGlnLysHisLeuGly                              115120125                                                                     CTTCATAAAGACTTTGATCAGTGGGACTGTCTGGTGGAGGGAGAGAAC430                           LeuHisLysAspPheAspGlnTrpAspCysLeuValGluGlyGluAsn                              130135140                                                                     GAGAACCTCATCCCGGGGTCACCCTTTGATGTAGTCACCCAGTCCCCG478                           GluAsnLeuIleProGlySerProPheAspValValThrGlnSerPro                              145150155                                                                     GCTCTGCAGAGTTCTCCCGGATCACACAACCAGAATTAG517                                    AlaLeuGlnSerSerProGlySerHisAsnGlnAsn                                          160165170                                                                     (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 580 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..580                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      CAAGTACTGATGGCTGCTGCAAGCTTTGGCGAACGTAAAATCCCTAAG48                            GlnValLeuMetAlaAlaAlaSerPheGlyGluArgLysIleProLys                              151015                                                                        GGAAATGGGCCTTATTCCGTTGGTTGTACAGACTTAATGTTTGATTAC96                            GlyAsnGlyProTyrSerValGlyCysThrAspLeuMetPheAspTyr                              202530                                                                        ACTAAAAAGGGCACCTTCTTGCGTTTATATTATCCATCCCAAGATGAT144                           ThrLysLysGlyThrPheLeuArgLeuTyrTyrProSerGlnAspAsp                              354045                                                                        GATCGCCTTGACACCCTTTGGATCCCAAATAAGGAGTATTTTTGGGGT192                           AspArgLeuAspThrLeuTrpIleProAsnLysGluTyrPheTrpGly                              505560                                                                        CTTAGCAAGTATCTTGGAAAACACTGGCTTATGGGCAACATTTTGAGT240                           LeuSerLysTyrLeuGlyLysHisTrpLeuMetGlyAsnIleLeuSer                              65707580                                                                      TTACTCTTTGGTTCAGTGACAACTCCTGCAAACTGGAATTCCCCTCTG288                           LeuLeuPheGlySerValThrThrProAlaAsnTrpAsnSerProLeu                              859095                                                                        AGGCCTGGTGAAAAATACCCACTTGTTGTTTTTTCTCATGGTCTTGGA336                           ArgProGlyGluLysTyrProLeuValValPheSerHisGlyLeuGly                              100105110                                                                     GCATTCAGGACAATTTATTCTGCTATTGGCATTGACCTGGCATCTCAT384                           AlaPheArgThrIleTyrSerAlaIleGlyIleAspLeuAlaSerHis                              115120125                                                                     GGGTTTATAGTTGCTGCTGTAGAACACAGAGATAGATCTGCATCTGCA432                           GlyPheIleValAlaAlaValGluHisArgAspArgSerAlaSerAla                              130135140                                                                     ACTTACTATTTCAAGAACCAATCTGCTGCAGAAATAGGGAAAAAGTCT480                           ThrTyrTyrPheLysAsnGlnSerAlaAlaGluIleGlyLysLysSer                              145150155160                                                                  TGGCTCTACCTTAGAACCCTGAAAGAAGAGGAGGAGATACATATACGA528                           TrpLeuTyrLeuArgThrLeuLysGluGluGluGluIleHisIleArg                              165170175                                                                     AATAAGCAGGTACGACAAAGAGCAAAAGAATGTTCCCAAGCTCTCAGT576                           AsnLysGlnValArgGlnArgAlaLysGluCysSerGlnAlaLeuSer                              180185190                                                                     CTGA580                                                                       Leu                                                                           (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      GlyXaaSerXaaGly                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      TATTCTAGAATTATGATACAAGTATTAATGGCTGCTGCAAG41                                   (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      ATTGATATCCTAATTGTATTTCTCTATTCCTG32                                            (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1335 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      ATGGTACCCCCAAAGCTGCACGTCCTGTTTTGTCTGTGTGGATGTCTCGCCGTCGTGTAC60                CCCTTCGATTGGCAGTATATCAACCCCGTGGCTCACATGAAGAGCAGCGCCTGGGTGAAT120               AAGATCCAGGTGCTCATGGCCGCACCAAGCTTCGGTCAGACCAAGATTCCTAGAGGCAAC180               GGCCCCTACAGCGTGGGCTGCACCGATCTGATGTTCGACCATACCAACAAAGGAACTTTT240               CTGAGACTGTACTACCCCAGCCAGGACAACGACAGACTGGATACTCTGTGGATCCCAAAT300               AAAGAATATTTTTGGGGTCTTAGCAAATTTCTTGGAACACACTGGCTTATGGGCAACATT360               TTGAGGTTACTCTTTGGTTCAATGACAACTCCTGCAAACTGGAATTCCCCTCTGAGGCCT420               GGTGAAAAATATCCACTTGTTGTTTTTTCTCATGGTCTTGGGGCATTCAGGACACTTTAT480               TCTGCTATTGGCATTGACCTGGCATCTCATGGGTTTATAGTTGCTGCTGTAGAACACAGA540               GATAGATCTGCATCTGCAACTTACTATTTCAAGGACCAATCTGCTGCAGAAATAGGGGAC600               AAGTCTTGGCTCTACCTTAGAACCCTGAAACAAGAGGAGGAGACACATATACGAAATGAG660               CAGGTACGGCAAAGAGCAAAAGAATGTTCCCAAGCTCTCAGTCTGATTCTTGACATTGAT720               CATGGAAAGCCAGTGAAGAATGCATTAGATTTAAAGTTTGATATGGAACAACTGAAGGAC780               TCTATTGATAGGGAAAAAATAGCAGTAATTGGACATTCTTTTGGTGGAGCAACGGTTATT840               CAGACTCTTAGTGAAGATCAGAGATTCAGATGTGGTATTGCCCTGGATGCATGGATGTTT900               CCACTGGGTGATGAAGTATATTCCAGAATTCCTCAGCCCCTCTTTTTTATCAACTCTGAA960               TATTTCCAATATCCTGCTAATATCATAAAAATGAAAAAATGCTACTCACCTGATAAAGAA1020              AGAAAGATGATTACAATCAGGGGTTCAGTCCACCAGAATTTTGCTGACTTCACTTTTGCA1080              ACTGGCAAAATAATTGGACACATGCTCAAATTAAAGGGAGACATAGATTCAAATGTAGCT1140              ATTGATCTTAGCAACAAAGCTTCATTAGCATTCTTACAAAAGCATTTAGGACTTCATAAA1200              GATTTTGATCAGTGGGACTGCTTGATTGAAGGAGATGATGAGAATCTTATTCCAGGGACC1260              AACATTAACACAACCAATCAACACATCATGTTACAGAACTCTTCAGGAATAGAGAAATAC1320              AATTAGGATTCTAGA1335                                                           (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 440 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      MetValProLeuLysLeuGlnAlaLeuPheCysLeuLeuCysCysLeu                              151015                                                                        ProTrpValHisProPheHisTrpGlnAspThrSerSerPheAspPhe                              202530                                                                        ArgProSerValMetPheHisLysLeuGlnSerValMetSerAlaAla                              354045                                                                        GlySerGlyHisSerLysIleProLysGlyAsnGlySerTyrProVal                              505560                                                                        GlyCysThrAspLeuMetPheGlyTyrGlyAsnGluSerValPheVal                              65707580                                                                      ArgLeuTyrTyrProAlaGlnAspGlnGlyArgLeuAspThrValTrp                              859095                                                                        IleProAsnLysGluTyrPheLeuGlyLeuSerIlePheLeuGlyThr                              100105110                                                                     ProSerIleValGlyAsnIleLeuHisLeuLeuTyrGlySerLeuThr                              115120125                                                                     ThrProAlaSerTrpAsnSerProLeuArgThrGlyGluLysTyrPro                              130135140                                                                     LeuIleValPheSerHisGlyLeuGlyAlaPheArgThrIleTyrSer                              145150155160                                                                  AlaIleGlyIleGlyLeuAlaSerAsnGlyPheIleValAlaThrVal                              165170175                                                                     GluHisArgAspArgSerAlaSerAlaThrTyrPhePheGluAspGln                              180185190                                                                     ValAlaAlaLysValGluAsnArgSerTrpLeuTyrLeuArgLysVal                              195200205                                                                     LysGlnGluGluSerGluSerValArgLysGluGlnValGlnGlnArg                              210215220                                                                     AlaIleGluCysSerArgAlaLeuSerAlaIleLeuAspIleGluHis                              225230235240                                                                  GlyAspProLysGluAsnValLeuGlySerAlaPheAspMetLysGln                              245250255                                                                     LeuLysAspAlaIleAspGluThrLysIleAlaLeuMetGlyHisSer                              260265270                                                                     PheGlyGlyAlaThrValLeuGlnAlaLeuSerGluAspGlnArgPhe                              275280285                                                                     ArgCysGlyValAlaLeuAspProTrpMetTyrProValAsnGluGlu                              290295300                                                                     LeuTyrSerArgThrLeuGlnProLeuLeuPheIleAsnSerAlaLys                              305310315320                                                                  PheGlnThrProLysAspIleAlaLysMetLysLysPheTyrGlnPro                              325330335                                                                     AspLysGluArgLysAsnAspTyrAsnGlnGlyLeuArgHisGlnAsn                              340345350                                                                     PheAspAspPheThrPheValThrGlyLysIleIleGlyAsnLysLeu                              355360365                                                                     ThrLeuLysGlyGluIleAspSerArgValAlaIleAspLeuThrAsn                              370375380                                                                     LysAlaSerMetAlaPheLeuGlnLysHisLeuGlyLeuGlnLysAsp                              385390395400                                                                  PheAspGlnTrpAspProLeuValGluGlyAspAspGluAsnLeuIle                              405410415                                                                     ProGlySerProPheAspAlaValThrGlnAlaProAlaGlnGlnHis                              420425430                                                                     SerProGlySerGlnThrGlnAsn                                                      435440                                                                        (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 444 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      MetLeuProProLysLeuHisAlaLeuPheCysLeuCysSerCysLeu                              151015                                                                        ThrLeuValHisProIleAspTrpGlnAspLeuAsnProValAlaHis                              202530                                                                        IleArgSerSerAlaTrpAlaAsnLysIleGlnAlaLeuMetAlaAla                              354045                                                                        AlaSerIleArgGlnSerArgIleProLysGlyAsnGlySerTyrSer                              505560                                                                        ValGlyCysThrAspLeuMetPheAspTyrThrAsnLysGlyThrPhe                              65707580                                                                      LeuArgLeuTyrTyrProSerGlnGluAspAspHisSerAspThrLeu                              859095                                                                        TrpIleProAsnLysGluTyrPhePheGlyLeuSerLysTyrLeuGly                              100105110                                                                     ThrProTrpLeuMetGlyLysIleLeuSerPhePhePheGlySerVal                              115120125                                                                     ThrThrProAlaAsnTrpAsnSerProLeuArgThrGlyGluLysTyr                              130135140                                                                     ProLeuIleValPheSerHisGlyLeuGlyAlaPheArgThrIleTyr                              145150155160                                                                  SerAlaIleGlyIleAspLeuAlaSerHisGlyPheIleValAlaAla                              165170175                                                                     IleGluHisArgAspGlySerAlaSerAlaThrTyrTyrPheLysAsp                              180185190                                                                     GlnSerAlaAlaGluIleGlyAsnLysSerTrpSerTyrLeuGlnGlu                              195200205                                                                     LeuLysProGlyAspGluGluIleHisValArgAsnGluGlnValGln                              210215220                                                                     LysArgAlaLysGluCysSerGlnAlaLeuAsnLeuIleLeuAspIle                              225230235240                                                                  AspHisGlyArgProIleLysAsnValLeuAspLeuGluPheAspVal                              245250255                                                                     GluGlnLeuLysAspSerIleAspArgAspLysIleAlaValIleGly                              260265270                                                                     HisSerPheGlyGlyAlaThrValLeuGlnAlaLeuSerGluAspGln                              275280285                                                                     ArgPheArgCysGlyIleAlaLeuAspAlaTrpMetLeuProLeuAsp                              290295300                                                                     AspAlaIleTyrSerArgIleProGlnProLeuPhePheIleAsnSer                              305310315320                                                                  GluArgPheGlnPheProGluAsnIleLysLysMetLysLysCysTyr                              325330335                                                                     SerProAspLysGluArgLysMetIleThrIleArgGlySerValHis                              340345350                                                                     GlnAsnPheAlaAspPheThrPheThrThrGlyLysIleValGlyTyr                              355360365                                                                     IlePheThrLeuLysGlyAspIleAspSerAsnValAlaIleAspLeu                              370375380                                                                     CysAsnLysAlaSerLeuAlaPheLeuGlnLysHisLeuGlyLeuArg                              385390395400                                                                  LysAspPheAspGlnTrpAspSerLeuIleGluGlyLysAspGluAsn                              405410415                                                                     LeuMetProGlyThrAsnIleAsnIleThrAsnGluHisAspThrLeu                              420425430                                                                     GlnAsnSerProGluAlaGluLysSerAsnLeuAsp                                          435440                                                                        (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 444 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      MetLeuProSerLysLeuHisAlaLeuPheCysLeuCysThrCysLeu                              151015                                                                        AlaLeuValTyrProPheAspTrpGlnAspLeuAsnProValAlaTyr                              202530                                                                        IleGluSerProAlaTrpValSerLysIleGlnAlaLeuMetAlaAla                              354045                                                                        AlaAsnIleGlyGlnSerLysIleProArgGlyAsnGlySerTyrSer                              505560                                                                        ValGlyCysThrAspLeuMetPheAspTyrThrAsnLysGlyThrPhe                              65707580                                                                      LeuArgLeuTyrTyrProSerGlnAspAspAspHisSerAspThrLeu                              859095                                                                        TrpIleProAsnLysGluTyrPheLeuGlyLeuSerLysPheLeuGly                              100105110                                                                     ThrHisTrpLeuValGlyLysIleMetGlyLeuPhePheGlySerMet                              115120125                                                                     ThrThrProAlaAlaTrpAsnAlaHisLeuArgThrGlyGluLysTyr                              130135140                                                                     ProLeuIleIlePheSerHisGlyLeuGlyAlaPheArgThrIleTyr                              145150155160                                                                  SerAlaIleGlyIleAspLeuAlaSerHisGlyPheIleValAlaAla                              165170175                                                                     ValGluHisArgAspGlySerAlaSerSerThrTyrTyrPheLysAsp                              180185190                                                                     GlnSerAlaValGluIleGlyAsnLysSerTrpLeuTyrLeuArgThr                              195200205                                                                     LeuLysArgGlyGluGluGluPheProLeuArgAsnGluGlnLeuArg                              210215220                                                                     GlnArgAlaLysGluCysSerGlnAlaLeuSerLeuIleLeuAspIle                              225230235240                                                                  AspHisGlyArgProValThrAsnValLeuAspLeuGluPheAspVal                              245250255                                                                     GluGlnLeuLysAspSerIleAspArgAspLysIleAlaIleIleGly                              260265270                                                                     HisSerPheGlyGlyAlaThrValIleGlnThrLeuSerGluAspGln                              275280285                                                                     ArgPheArgCysGlyIleAlaLeuAspAlaTrpMetPheProValGly                              290295300                                                                     AspGluValTyrSerArgIleProGlnProLeuPhePheIleAsnSer                              305310315320                                                                  GluArgPheGlnTyrProSerAsnIleIleArgMetLysLysCysPhe                              325330335                                                                     LeuProAspArgGluArgLysMetIleThrIleArgGlySerValHis                              340345350                                                                     GlnAsnPheValAspPheThrPheAlaThrSerLysIleIleGlyTyr                              355360365                                                                     LeuPheThrLeuLysGlyAspIleAspSerAsnValAlaIleSerLeu                              370375380                                                                     SerAsnLysAlaSerLeuAlaPheLeuGlnLysHisLeuGlyLeuGln                              385390395400                                                                  LysAspPheAspGlnTrpAspSerLeuValGluGlyGluAspHisAsn                              405410415                                                                     LeuIleProGlyThrAsnIleAsnThrThrAsnHisGlnAlaIleLeu                              420425430                                                                     GlnAsnSerThrGlyIleGluArgProAsnLeuAsp                                          435440                                                                        (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 422 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      MetAlaSerLeuTrpValArgAlaArgArgValPheMetLysSerArg                              151015                                                                        AlaSerGlyPheSerAlaLysAlaAlaThrGluMetGlySerGlyGly                              202530                                                                        AlaGluLysGlyTyrArgIleProAlaGlyLysGlyProHisAlaVal                              354045                                                                        GlyCysThrAspLeuMetThrGlyAspAlaAlaGluGlySerPheLeu                              505560                                                                        ArgLeuTyrTyrLeuSerCysAspAspThrAspThrGluGluThrPro                              65707580                                                                      TrpIleProAspLysGluTyrTyrGlnGlyLeuSerAspPheLeuAsn                              859095                                                                        ValTyrArgAlaLeuGlyGluArgLeuPheGlnTyrTyrValGlySer                              100105110                                                                     ValThrCysProAlaLysSerAsnAlaAlaPheLysProGlyGluLys                              115120125                                                                     TyrProLeuLeuValPheSerHisGlyLeuGlyAlaPheArgThrIle                              130135140                                                                     TyrSerAlaIleCysIleGluMetAlaSerGlnGlyPheLeuValAla                              145150155160                                                                  AlaValGluHisArgAspGluSerAlaSerAlaThrTyrPheCysLys                              165170175                                                                     LysLysAlaAspSerGluProGluGluAspGlnThrSerGlyValGlu                              180185190                                                                     LysGluTrpIleTyrTyrArgLysLeuArgAlaGlyGluGluGluArg                              195200205                                                                     CysLeuArgHisLysGlnValGlnGlnArgAlaGlnGluCysIleLys                              210215220                                                                     AlaLeuAsnLeuIleLeuLysIleSerSerGlyGluGluValMetAsn                              225230235240                                                                  ValLeuAsnSerAspPheAspTrpAsnHisLeuLysAspSerValAsp                              245250255                                                                     ThrSerArgIleAlaValMetGlyHisSerPheGlyGlyAlaThrVal                              260265270                                                                     IleGluSerLeuSerLysGluIleArgPheArgCysGlyIleAlaLeu                              275280285                                                                     AspAlaTrpMetLeuProValGlyAspAspThrTyrGlnSerSerVal                              290295300                                                                     GlnGlnProLeuLeuPheIleAsnSerGluLysPheGlnTrpAlaAla                              305310315320                                                                  AsnIleLeuLysMetLysLysLeuSerSerAsnAspThrAsnLysLys                              325330335                                                                     MetIleThrIleLysGlySerValHisGlnSerPheProAspPheThr                              340345350                                                                     PheValSerGlyGluIleIleGlyLysPhePheLysLeuLysGlyGlu                              355360365                                                                     IleAspProAsnGluAlaIleAspIleCysAsnHisAlaSerLeuAla                              370375380                                                                     PheLeuGlnLysHisLeuSerLeuLysArgAspPheAspLysTrpAsp                              385390395400                                                                  SerLeuValAspGlyIleGlyProAsnValIleSerGlyThrAsnIle                              405410415                                                                     AspLeuSerProThrGlu                                                            420                                                                           (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 171 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      GlyHisSerPheGlyGlyAlaThrValPheGlnAlaLeuSerGluAsp                              151015                                                                        GlnArgPheArgCysGlyIleAlaLeuAspProTrpMetPheProVal                              202530                                                                        SerGluGluLeuTyrSerArgValProGlnProLeuPhePheIleAsn                              354045                                                                        SerAlaGluPheGlnThrProLysAspIleAlaLysMetLysAsnPhe                              505560                                                                        TyrGlnProAspLysGluArgLysMetIleThrIleLysGlySerVal                              65707580                                                                      HisGlnAsnPheAlaAspGlyThrPheValThrGlyLysIleIleGly                              859095                                                                        AsnLysLeuSerLeuLysGlyAspIleAspSerArgValAlaIleAsp                              100105110                                                                     LeuThrAsnLysAlaSerLeuAlaPheLeuGlnLysHisLeuGlyLeu                              115120125                                                                     HisLysAspPheAspGlnTrpAspCysLeuValGluGlyGluAsnGlu                              130135140                                                                     AsnLeuIleProGlySerProPheAspValValThrGlnSerProAla                              145150155160                                                                  LeuGlnSerSerProGlySerHisAsnGlnAsn                                             165170                                                                        (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 193 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      GlnValLeuMetAlaAlaAlaSerPheGlyGluArgLysIleProLys                              151015                                                                        GlyAsnGlyProTyrSerValGlyCysThrAspLeuMetPheAspTyr                              202530                                                                        ThrLysLysGlyThrPheLeuArgLeuTyrTyrProSerGlnAspAsp                              354045                                                                        AspArgLeuAspThrLeuTrpIleProAsnLysGluTyrPheTrpGly                              505560                                                                        LeuSerLysTyrLeuGlyLysHisTrpLeuMetGlyAsnIleLeuSer                              65707580                                                                      LeuLeuPheGlySerValThrThrProAlaAsnTrpAsnSerProLeu                              859095                                                                        ArgProGlyGluLysTyrProLeuValValPheSerHisGlyLeuGly                              100105110                                                                     AlaPheArgThrIleTyrSerAlaIleGlyIleAspLeuAlaSerHis                              115120125                                                                     GlyPheIleValAlaAlaValGluHisArgAspArgSerAlaSerAla                              130135140                                                                     ThrTyrTyrPheLysAsnGlnSerAlaAlaGluIleGlyLysLysSer                              145150155160                                                                  TrpLeuTyrLeuArgThrLeuLysGluGluGluGluIleHisIleArg                              165170175                                                                     AsnLysGlnValArgGlnArgAlaLysGluCysSerGlnAlaLeuSer                              180185190                                                                     Leu                                                                           __________________________________________________________________________

We claim:
 1. An antibody which specifically binds to humanplatelet-activating factor acetylhydrolase (PAF-AH) comprising thePAF-AH amino acid sequence set out in SEQ ID NO:8.
 2. The monoclonalantibody produced by hybridoma 90G11D (ATCC HB 11724).
 3. The monoclonalantibody produced by hybridoma 90F2D (ATCC HB 11725).
 4. The monoclonalantibody produced by hybridoma 143A (ATCC HB 11900).
 5. A hybridoma cellline producing a monoclonal antibody according to claim
 1. 6. Thehybridoma cell line 90G11D (ATCC HB 11724).
 7. The hybridoma cell line90F2D (ATCC HB 11725).
 8. The hybridoma cell line 143A (ATCC HB 11900).9. A humanized antibody which specifically binds to human PAF-AHcomprising the PAF-AH amino acid sequence set out in SEQ ID NO:8.
 10. Amonoclonal antibody which specifically binds to murine PAF-AH comprisingthe PAF-AH amino acid sequence set out in SEQ ID NO:31.
 11. A monoclonalantibody which specifically binds to canine PAF-AH comprising the PAF-AHamino acid sequence set out in SEQ ID NO:32.
 12. A monoclonal antibodywhich specifically binds to bovine PAF-AH comprising the PAF-AH aminoacid sequence set out in SEQ ID NO:33.
 13. A monoclonal antibody whichspecifically binds to chicken PAF-AH comprising the PAF-AH amino acidsequence set out in SEQ ID NO:34.
 14. A monoclonal antibody whichspecifically binds to rodent PAF-AH comprising the PAF-AH amino acidsequence set out in SEQ ID NO:35.
 15. A monoclonal antibody whichspecifically binds to macaque PAF-AH comprising the PAF-AH amino acidsequence set out in SEQ ID NO:36.